Particulate delivery systems and methods of use

ABSTRACT

Compositions and methods are provided for the administration of particulates comprising at least one bioactive agent which, in selected embodiments, may comprise and immunoactive agent. In this respect, the invention provides for both topical and systemic delivery of the bioactive agent using, for example, the respiratory, gastrointestinal or urogenital tracts. The particulates may be in the form of dry powders or combined with a non-aqueous suspension medium to provide stabilized dispersions. In preferred embodiments, the disclosed compositions will be used in conjunction with inhalation devices such as metered dose inhalers, dry powder inhalers, atomizers or nebulizers for targeted delivery of the agent to mucosal surfaces.

This application is a 371 of PCT/US 99/06855 filed Mar. 31, 1999.

FIELD OF THE INVENTION

The present invention generally relates to compositions and methods forthe administration of particulates comprising at least one bioactiveagent which, in selected embodiments, may comprise an immunoactiveagent. In this respect, the invention provides for both topical andsystemic delivery of the bioactive agent using, for example, therespiratory, gastrointestinal or urogenital tracts. In particularlypreferred embodiments, the disclosed compositions will be used inconjunction with inhalation devices such as metered dose inhalers, drypowder inhalers, atomizers or nebulizers for targeted delivery tomucosal surfaces.

BACKGROUND OF THE INVENTION

Vertebrates possess the ability to mount an immune response as a defenseagainst pathogens from the environment as well as against aberrantcells, such as tumor cells, which develop internally. This can take theform of innate or passive immunity, which is mediated by neutrophils andcells of the monocyte/macrophage lineage, or the form of acquired oractive immunity mediated by lymphocytes against a specific antigenicsequence. Active immune responses can themselves be further subdividedinto two arms, the humoral response which entails the production ofspecific antibodies which serve to neutralize antigens exposed to thesystemic circulation and aid in their uptake by professional phagocyticcells, and the cellular arm which is required for recognition ofinfected or aberrant cells within the body.

In both cases the specific response is triggered by the intracellularprocessing of antigen. When the antigen is processed through thecytoplasmic route, the resultant peptides are bound to nascent MHC classI molecules which facilitates appropriate presentation to effectorT-cells. MHC class I presentation favors recognition by cytotoxic Tlymphocytes. In contrast, intracellular processing via the endocyticroute results in presentation on MHC class II molecules which favors Thelper responses involved in stimulation of the humoral arm. The goal ofvaccination is to prime both responses and generate memory T cells, suchthat the immune system is primed to react to a pathogenic infection.Such a response is promoted by the coadministration of signals thatpromote costimulatory molecule expression, so called “adjuvants.”Engagement of both the humoral and cellular immune responses leads tobroad based immunity and is the preferred goal for intracellularpathogens. The absence of appropriate costimulatory molecule expressioncan lead to a state of T cell unresponsiveness.

In this regard, modulation of an immune response can take one of twodirections; either to elicit an immune response directed against aforeign pathogenic agent or antigen thereof, or to suppress aninappropriate reaction mounted against a self-epitope that leads tochronic inflammation. Such chronic reactions against self-epitopes areassociated with various autoimmune diseases such as diabetes, typicallytype I, multiple sclerosis, rheumatoid arthritis or lupuserythrematosis. In either case, the active agent frequently takes theform of a relatively complex peptide, protein, RNA or DNA-based entityor other macromolecular structure rather than small chemical entitiestypical of classical pharmaceutical agents. These complex bioactiveagents generally exhibit poor bioavailability when administered orally,and therefore have traditionally been administered by invasiveparenteral injection. Recently however, it has been suggested thatrelatively large biomolecules may be delivered via mucosal routes, e.g.by inhalation. Delivery of these agents into systemic circulationthrough inhalation is particularly attractive since administration viathe respiratory mucosa bypasses the digestive enzymes of the GI tract.Furthermore, it offers the potential for increased bioavailability forpeptides and proteins because of the large surface area available forexchange with systemic circulation. While the molecular weight cut-offfor oral bioavailability is generally regarded to be in the range of 500Daltons, peptide hormones or analogues of larger molecular weight (e.g.,1.8 kD desmopressin, 5.8 kD insulin, 9.5 kD parathyroid hormone), havebeen shown to be absorbed across the nasal or pulmonary mucosa intactinto the systemic circulation.

Besides allowing for the effective delivery of protein, peptide, viraland DNA formulations without degradation, targeted delivery to themucosal surface itself may offer a benefit if it elicits a local immuneresponse within the MALT (mucosa-associated lymphoid tissue) lymphoidsystem. Mucosal vaccination is of particular interest for vaccinesdesigned against pathogens whose port of entry is typically at one ofthe mucosal surfaces interfacing the body with the external environment.The MALT lymphoid system resides within the lamina propria of themucosa. When foreign antigen is presented to local dendritic cells,there is a local amplification and maturation of B-cell precursors,which produce IgA and IgM antibodies in addition to the IgG antibodiestypically induced by systemic delivery of antigen. The former aresecreted through specialized transport receptors by a process known astranscytosis across the mucosal surface into the lumen. There, theyprovide a first line of defense against invading pathogens at themucosal surface. Recent evidence indicates that, in addition to bindingpathogenic antigens, the resultant formation of immune complexes may inand of itself inhibit viral transmission occurring via the transcytoticroute. By priming this first line immune response to antigens derivedfrom pathogens, mucosal immunization should greatly enhance theefficiency with which the organism first intercepts an invadingpathogen.

Several previous attempts have been made to exploit this uptakemechanism and provide for the effective delivery of peptides orproteins. For example, U.S. Pat. No. 5,756,104 describes the use ofliposome formulations for intranasal vaccine formulations. Theseformulations appear to comprise aqueous carriers having liposomes andfree antigenic material dispersed therein. While the compositions werefound to elicit an immune response, they appear to be extremely labileand susceptible to degradation over time. In a practical sense this is asubstantial drawback.

Attempts to overcome such limitations and further increase deliveryefficiency have resulted in the development of dry powders for theadministration of relatively large biomolecules. Unfortunately,conventional powdered preparations (i.e. micronized) often fail toprovide accurate, reproducible dosing over extended periods. In part,this is because the powders tend to aggregate due to hydrophobic orelectrostatic interactions between the fine particles. Such cohesion maybe partially overcome through the use of larger carrier particles (i.e.lactose) to inhibit aggregation. However, these larger particles andassociated drug often fail to reach the targeted cells resulting inuneven delivery profiles. Further, crude mixtures comprising carriermolecules provide little, if any, protection for the incorporatedbiomolecule. Accordingly, as with the aqueous compositions describedabove, such preparations are subject to degradation and loss of activityover time.

More recently, improved formulation methods have been undertaken inorder to overcome the limitations associated with conventional prior artpowders and aqueous preparations. In this regard, U.S. patentapplications Ser. Nos. 09/218,209 and 09/219,736, incorporated herein byreference, describe methods and processes for generating preparationscomprising bioactive agents in microparticulate form. The resultantpowders, which preferably exhibit a hollow, porous morphology, aresuitable for use in inhalation devices such as dry powder inhalers(DPIs) or, when suspended in a nonaqueous liquid (i.e. ahydrofluoroalkane or fluorocarbon), metered dose inhalers (MDIs) andnebulizers. Moreover, the mild conditions used during the formulationprocess support retention of biological activity making the preparationsparticularly compatible for use with proteins and peptides as well asmore complex macromolecular structures such as viruses. Additionally,since the resultant powders have very low residual water content, whichcan be further maintained by formulation in short-chain fluorocarbons orfluorochemicals such as propellants or the longer chain fluorochemicalssuch as perfluorooctyl bromide (PFOB), these formulations provide astable means for storage of labile bioactive agents.

Besides enhanced stability, the preferred hollow, porous morphology ofthe microparticulates provides aerodynamic characteristics that areparticularly compatible with inhalation therapies. Further, theparticulate characteristics allows for the formation of exceptionallystable dispersions and makes them especially compatible withhydrofluoroalkane propellants such as HFA-134a as well as otherfluorocarbon liquid vehicles like PFOB. Thus, whether used in a dry formor as a nonaqueous dispersion, the microparticulates provide for gooddose reproducibility, excellent plume characteristics (a measure of theuniformity of a propellant or dry powder spray) and a high percentage ofthe dose delivered as the respirable fraction (as opposed to depositionin the device or throat). These properties suggest that the disclosedmicroparticles offer substantial theoretical advantages as far asdelivery deep into the lung. Such deep deposition is preferred wheredelivery into the systemic circulation is desired since uptake of largemacromolecules like proteins and peptides is optimal at the level of thealveoli.

While the use of such microparticulate preparations is a substantialimprovement over conventional prior art delivery methods, there stillremains a need to provide for the targeted delivery of bioactive,immunomodulating or immunoactive agents that results in an enhancedphysiological response.

Accordingly, it is an object of the present invention to providecompositions, systems and methods that provide for the generation of anenhanced immune response.

It is another object to provide for the effective delivery ofimmunoactive agents, including vaccines and immunomodulating agents, tothe mucosal surfaces of a patient in need thereof.

It is yet a further object of the present invention to provide vaccineor other bioactive formulations that do not require refrigeration orfreezing to maintain activity.

It is still a further object of the present invention to provide for theestablishment of passive and active immunity via inhalation therapies.

It is yet another object of the present invention to provide for stablepreparations of immunoactive agents that may be used to confer immunityor down regulate the immune system of a patient in need thereof.

SUMMARY OF THE INVENTION

These and other objects are provided for by the invention disclosed andclaimed herein. To that end, the methods and associated compositions ofthe present invention allow, in a broad aspect, for the improveddelivery of bioactive agents to selected target sites in a powdered orparticulate form. More particularly, it has been surprisingly been foundthat the disclosed methods and compositions may be used to enhance orincrease the activity of an incorporated bioactive agent, whichpreferably comprises an immunoactive agent, following administration. Inthis regard, the vaccines of the instant invention appear to exhibit an“adjuvant effect” that may provoke an enhanced immune response an orderof magnitude or more greater than that provoked by a comparable priorart vaccine formulation. Besides this unexpected improvement in potency,relatively gentle formulation techniques may be combined withparticulate morphology and composition to protect and enhance theactivity of any incorporated agents. This allows for the formation ofrelatively efficacious preparations that retain their biologicalactivity without the need for refrigeration or freezing. Further, unlikeprior art powders or dispersions for drug delivery, the presentinvention preferably employs novel techniques to reduce attractiveforces between the particles, resulting in improved flowability anddispersibility. When these powders are incorporated in a nonaqueoussuspension medium (e.g. a liquid fluorochemical) these samecharacteristics provide for reduced flocculation, sedimentation orcreaming that may further reduce the rate of agent degradation. Finally,administration of the disclosed particulates or dispersions to selectedtarget sites such as mucosal surfaces may further serve to optimize orenhance bioactivity. As such, the dispersions or powders of the presentinvention may be used to effectively deliver bioactive agents inconjunction with metered dose inhalers, dry powder inhalers, atomizers,aerosolizers, nasal pumps, spray bottles, nebulizers or liquid doseinstillation (LDI) techniques.

A particularly beneficial feature of the disclosed particulateformulation technology is that a wide range of bioactive structures canbe incorporated in the stabilized dispersions or powders irrespective oftheir hydrophobicity or hydrophilicity. In preferred embodiments, thebioactive powders will be produced using relatively mild spray dryingmethodology. Due to such compatible particulate formulation techniques,larger, more labile biomolecules such as peptides, proteins or geneticmaterial may readily be incorporated in the disclosed compositionswithout adverse effects or undue loss of activity. These sameformulation techniques and resulting particulates further provide forthe incorporation and delivery of relatively high doses (ca. 10 mg) ofbioactive agents using conventional administration techniques andsystems. Thus, whether administered in the form of a dry powder orstabilized dispersion, the novel particulate fabrication techniques andenhanced response afforded by the disclosed preparations lead to theeffective delivery of bioactive agents to targeted sites such as themucosa.

In connection with the present invention, the term “bioactive agent”refers to any active peptide or protein, such as a hormone, cytokine orchemokine or an immunoactive agent. That is, while the disclosedcompositions and methods are compatible with almost any bioactive agent,they have been discovered to be surprisingly effective for the deliveryor administration of immunoactive agents designed to modulate immuneresponses such as, for example, eliciting an immune response to aforeign antigen or pathogen or down regulating an active immunereaction. Accordingly, as used herein, the terms “immunoactive agents,”or “immunologically active agents,” will comprise any molecule that maybe used to elicit a physiological or immune response or modulatepre-existing responses in a subject. Such immunoactive agents orbiologics may comprise peptides, polypeptides, proteins, carbohydrates,genetic material including DNA, RNA and antisense constructs, as well asmicrobes including viruses, phages and bacteria.

In addition, molecules that may function as cofactors, potentiators orpenetration enhancers can be readily co-formulated in the particulatesdescribed herein. Those skilled in the art will appreciate that anycompound which acts to improve the uptake, presentation orbioavailability may function as a potentiator or penetration enhancer inaccordance with the teachings herein. For instance, compounds that canalter or increase the membrane permeability of a cell may function aspotentiators or penetration enhancers. Exemplary potentiators orpenetration enhancers may include chelating agents (e.g. EDTA, citricacid), detergents or surfactants (e.g. 9-lauryl ether), fatty acids(e.g. oleic acid) and bile salts (e.g. sodium glycocholate).Particularly preferred penetration enhancers comprise relatively shortchain phospholipids having chain lengths of less than about 10 carbons.As with the bioactive agents, and as will be discussed in more detailbelow, the selected potentiators or penetration enhancers may beincorporated in, or associated with, particulates in varyingconcentrations.

With regard to the particulates, microparticulates or perforatedmicrostructures of the present invention, those skilled in the art willappreciate that they may be formed of any biocompatible materialproviding the desired physical characteristics or morphology. In thisrespect, perforated microstructures will preferably comprise pores,voids, defects or other interstitial spaces that act to reduceattractive forces by minimizing surface interactions and decreasingshear forces. This morphology acts to reduce aggregation and improvedispersability. Yet, given these constraints, it will be appreciatedthat any biocompatible material or configuration may be used to form themicrostructure matrix. As to the selected materials, it is desirablethat the microstructure incorporates at least one surfactant which, inpreferred embodiments, will act as a penetration enhancer. Preferably,this surfactant will comprise a phospholipid or other surfactant oramphiphile approved for pharmaceutical use. Similarly, it is preferredthat the microstructures incorporate at least one bioactive agent orbiologic. As to the configuration, selected embodiments of the inventioncomprise spray dried, hollow microspheres having a relatively thinporous wall defining a large internal void, although, other voidcontaining or perforated structures are contemplated as well.

It has unexpectedly been found that the use of hollow and/or porousperforated microstructures may substantially reduce attractive molecularforces, such as van der Waals forces, which dominate prior art powderedpreparations and dispersions. In this respect, the powdered compositionstypically have relatively low bulk densities that contribute to theflowability of the preparations while providing the desiredcharacteristics for inhalation therapies. More particularly, the use ofrelatively low density perforated (or porous) microstructures ormicroparticulates significantly reduces attractive forces between theparticles thereby lowering the shear forces required to achieveflowability of the resulting powders. The relatively low density of theperforated microstructures also provides for superior aerodynamicperformance when used in inhalation therapy. In dispersions, thephysical characteristics of these powders provide for the formation ofstable preparations. Moreover, by selecting dispersion components inaccordance with the teachings herein, interparticle attractive forcesmay further be reduced to provide formulations or preparations havingenhanced stability.

While preferred embodiments of the invention comprise perforatedmicrostructures or porous particulates, relatively nonporous or solidparticulates may also be used to prepare powders or dispersions that arecompatible with the teachings herein. That is, powders or dispersionscomprising suspensions of relatively nonporous or solid particulates arealso contemplated as being within the scope of the present invention. Inthis respect, such relatively nonporous particulates may comprisemicronized particles, milled particles or nanocrystals. Accordingly, asused herein the term “particulate” shall be interpreted broadly and heldto comprise particles of any porosity and or density, including bothperforated microstructures and relatively nonporous particles.

As previously alluded to, the disclosed powders may be dispersed in anappropriate nonaqueous suspension medium to provide stabilizeddispersions comprising a selected bioactive agent. Such dispersions areparticularly useful in metered dose inhalers, atomizers nasal pumps,spray bottles and nebulizers. Other embodiments of the inventioncomprise stabilized dispersions that may be administered directly to thelung or nasal cavity using direct instillation techniques. In any case,particularly preferred suspension mediums comprise fluorochemicals (i.e.perfluorocarbons or fluorocarbons) that are liquid at room temperatureor fluorinated propellants (i.e. hydrofluoroalkanes orchlorofluorocarbons). Because of their beneficial wettingcharacteristics, some fluorochemicals may be able to provide for thedispersion of particles deeper into the lung or other mucosal surface,thereby improving systemic delivery. Moreover, such suspension mediatend to be anhydrous thereby retarding hydrolytic degradation of theincorporated bioactive agents. Finally, fluorochemicals are generallybacteriostatic thus decreasing the potential for microbial growth andassociated proteolytic decay in compatible preparations.

With regard to the delivery of the disclosed powders or stabilizeddispersions, another aspect of the present invention is directed toinhalation systems for the administration of one or more bioactiveagents or biologics to a patient. As alluded to above, exemplaryinhalation devices compatible with the present invention may comprise anatomizer, a nasal pump, a sprayer or spray bottle, a dry powder inhaler,a metered dose inhaler or a nebulizer. In preferred embodiments, theseinhalation systems will deliver the bioactive agent to the desiredphysiological site (e.g. a mucosal surface) as an aerosol. For thepurposes of the instant application the term “aerosolized” shall be heldto mean a gaseous suspension of fine solid or liquid particles unlessotherwise dictated by contextual restraints. That is, an aerosol oraerosolized medicament may be generated, for example, by a dry powderinhaler, a metered dose inhaler, an atomizer, a spray bottle or anebulizer. Of course, as explained in more detail below, thecompositions of the present invention may also be delivered directly(e.g. by conventional injection or needleless injection) or using suchtechniques as liquid dose instillation. In especially preferredembodiments the compositions of the present invention are contacted witha mucosal surface (e.g. via inhalation) to elicit both mucosal andsystemic immunity.

While the powders or stabilized dispersions of the present invention areparticularly suitable for administration of bioactive agents to mucosalsurfaces, it will be appreciated that they may also be used for thelocalized or systemic administration of compounds to any location of thebody. Accordingly, it should be emphasized that, in preferredembodiments, the formulations may be administered using a number ofdifferent routes including, but not limited to, the gastrointestinaltract, the respiratory tract, topically, intramuscularly, parenterally,intradermally, transdermally, intraperitoneally, nasally, vaginally,rectally, aurally, buccally, orally or ocularly. In this respect thoseskilled in the art will appreciate that the selected route ofadministration will largely be determined by the choice of bioactiveagent and the desired response of the subject.

Other objects, features and advantages of the present invention will beapparent to those skilled in the art from a consideration of thefollowing detailed description of preferred exemplary embodimentsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of levels of functional HA peptide(residues 110-120 of the hemagglutinin of the influenza virus) followingformulation in microstructures according to the present invention;

FIG. 2 illustrates the fact that antigens formulated in microstructuresdo not require intracellular processing to activate T cells;

FIG. 3 graphically compares the plasma concentration of HA peptidedelivered using nasally administered microparticulates and intravenousinjection;

FIG. 4 depicts calibration curves for human IgG formulated inmicroparticulates as described in the instant application along withselected controls;

FIGS. 5A and 5B graphically illustrate release kinetics for IgGformulated microparticulates and HA peptide formulated microparticulatesrespectively;

FIGS. 6A and 6B show the persistence of IgG in the plasma followingintratracheal and nasal administration using formulatedmicroparticulates;

FIGS. 7A and 7B show, respectively, systemic and localized antibodyresponses to IgG administered intratracheally as formulatedmicroparticulates in accordance with the present invention;

FIG. 8 graphically illustrates levels of cytokines indicative of a Tcell response following intratracheal administration of IgGmicroparticulates to mice;

FIG. 9 depicts murine antibody response to IgG microparticulatesadministered intranasally;

FIGS. 10A and 10B present murine antibody titers at 7 and 14 daysrespectively following intraperitoneal administration of IgGmicroparticulates;

FIGS. 11A, 11B and 11C respectively illustrate T cell responses tomicroparticulate formulated virus, viral control and infectious titer ofboth formulated and unformulated virus;

FIG. 12 depicts murine antibody responses to microparticulate formulatedlive and killed influenza virus at 7 and 14 days following intranasaladministration;

FIGS. 13A, 13B and 13C show, respectively, murine levels of factorsindicative of a T cell response following intranasal inoculation ofviral microparticulates or live virus or killed virus along with controlantigens;

FIGS. 14A and 14B respectively illustrate viral shedding and body weightvariation in mice intranasally inoculated with microparticulatescomprising both live and killed virus; and

FIG. 15 presents results of an in vitro Andersen cascade impactor studyshowing efficient delivery of formulated microspheres comprising bovinegamma globulin from a metered dose inhaler.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS A. Introduction

While the present invention may be embodied in many different forms,disclosed herein are specific illustrative embodiments thereof thatexemplify the principles of the invention. It should be emphasized thatthe present invention is not limited to the specific embodiments asillustrated.

As discussed above, the present invention provides methods, systems andcompositions comprising powders or microparticulates that mayadvantageously be used for the delivery of bioactive agents. Preferablythe bioactive agent will comprise active peptides or proteins or animmunoactive agent. In the context of the present invention,immunoactive agents may comprise any molecule that may be used to elicitan immune response or modulate pre-existing responses such as vaccines,immunoglobulins or autoantigens. Those skilled in the art willappreciate that the disclosed powders may advantageously be used todeliver bioactive agents in a dry state (e.g. with a DPI or gas drivenpowder injector) or in the form of a stabilized dispersion (e.g. with anatomizer, spray bottle, MDI, LDI, needleless injector, syringe, nasalpump or nebulizer). In particularly preferred embodiments, the powdersor microparticulates will comprise perforated microstructures which, asdisclosed herein, comprise a structural matrix that exhibits, defines orcomprises voids, pores, defects, hollows, spaces, interstitial spaces,apertures, perforations or holes. These perforated microstructurepowders have aerodynamic characteristics that make them particularlyuseful for inhalation therapy and exhibit morphologies that allow forthe formation of stabilized dispersions in propellants or nonaqueousdelivery vehicles. More generally, the relatively mild conditionsemployed during the formation of the disclosed bioactive powders andadvantages associated with compatible delivery methods allow for theefficient administration of comparatively fragile biologic agents.

While not wishing to be bound by any particular theory, it is believedthat the relatively gentle methods used to form, store and administerthe disclosed compositions provide for the effective retention ofbiological activity in generally unstable agents. In this respect,preferred formulations do not require refrigeration to maintain theiractivity. Moreover, selection of appropriate compounds for use in thedisclosed powders and delivery to selected physiological sites (e.g.mucosal surfaces) may promote the uptake of the incorporated agent oragents as well as enhancing the activity thereof. In addition, thecompositions and/or delivery techniques of the present invention appearto generate an unexpected “adjuvant effect” that may provide for anenhanced immune response or bioactivity following administration of theselected agent. More specifically, as will be discussed below and seenin the Examples, the present invention may be used to elicit an immuneresponse comparable to that achieved by administering an antigen incomplete Freund's adjuvant (i.e. an order of magnitude or more higherthan conventional pharmaceutical formulations). Accordingly, the presentinvention provides for the effective delivery of active peptides,proteins, genetic material, or pathogenic particles (either live orinactivated) to induce active localized or systemic immunization or toachieve passive immunization, immune modulation, hormonal regulation orgene therapy.

B. Bioactive Agents

In a broad aspect, the powdered or microparticulate compositions of thepresent invention, including dispersions incorporating such powders,will preferably comprise at least one bioactive agent. As used herein,the term “bioactive agent” shall be held to comprise any active peptideor protein or any immunoactive agent. With respect to the latter,particularly preferred embodiments of the present invention willcomprise an immunoactive agent designed capable of modulating an immuneresponse. In accordance with the teachings herein, modulation of asubject's immune response shall comprise eliciting a response against apotential pathogenic infection or foreign antigen, stimulating anexisting immune response, inducing localized or systemic passiveimmunity or suppressing an autoimmune response or allergenic response.For the purposes of the instant application the terms “bioactive agent”or immunoactive agent” shall be broadly construed to comprise anymolecule or organism, or analog, homologue or derivative thereof, thatprovides a desired physiological or immune response in a subject. Itwill be appreciated that the term “bioactive agent” shall be heldinclusive of the term “immunoactive agent” and its equivalents unlessotherwise dictated by contextual restraints. Exemplary bioactive agentsthat may be used in conjunction with the invention comprise peptides,polypeptides, proteins, fusion or chimeric proteins, immunoglobulins,genetic material including DNA, RNA, recombinant and antisenseconstructs, microbes including viruses, phages, bacterial carbohydratesand bacteria as well as smaller molecules that may function aspotentiators, cofactors or penetration enhancers. The bioactivecompositions according to the present invention find use as vaccines,immunomodulators, effectors or replicons for gene therapy applications.

It will be appreciated that the powders or microparticulate compositionsof the present invention may exclusively comprise one or more bioactiveagent(s) (i.e. up to 100% w/w). However, in selected embodiments theperforated microstructures may incorporate much less bioactive agentdepending on the activity thereof. Accordingly, for highly activematerials the particulates, microparticulates or perforatedmicrostructures may incorporate as little as 0.001% by weight although aconcentration of greater than about 0.1% w/w is preferred. Otherembodiments of the invention may comprise greater than about 5%, 10%,15%, 20%, 25%, 30% or even 40% w/w active or bioactive agent orbiologic. Still more preferably the disclosed powders may comprisegreater than about 50%, 60%, 70%, 75%, 80% or even 90% w/w of abioactive agent. The precise amount of bioactive agent incorporated inthe powders or perforated microstructures of the present invention isdependent upon the agent of choice, the required dose, method ofadministration and the form of the agent actually used. Those skilled inthe art will appreciate that such determinations may be made by usingwell-known pharmacological techniques in combination with the teachingsof the present invention.

With regard to pharmaceutical preparations, any bioactive agent that maybe formulated in the disclosed powders or perforated microstructures forthe purpose of eliciting a physiological response, including an immuneresponse, is expressly held to be within the scope of the presentinvention. In accordance with the teachings herein the selectedbioactive agent(s) may be associated with, or incorporated in, thepowders or perforated microstructures in any form that provides thedesired efficacy and is compatible with the chosen productiontechniques. As used herein, the terms “associate” or “associating” meanthat the particulate, microparticulate, structural matrix or perforatedmicrostructure may comprise, incorporate, adsorb, absorb, be coated withor be formed by the bioactive agent. Where appropriate, the agent may beused in the form of salts (e.g. alkali metal or amine salts or as acidaddition salts) or as esters or as solvates (hydrates). In this regardthe form of the bioactive agent may be selected to optimize the activityand/or stability of the compound and/or to minimize the solubility ofthe agent in the suspension medium and/or to minimize particleaggregation.

At least to some extent, the advantages provided by the instantinvention reside in the unique formulation, storage and delivery aspectsafforded by the disclosed powders and dispersions. In this respect, andas will be discussed in more detail below, the conditions under whichthe disclosed powders or perforated microstructures may be formed arerelatively mild. That is, particulates comprising bioactive agents maybe formed according to the present invention without subjecting theactive compound or agent to extreme physical or chemical conditions.This is of extreme importance with regard to relatively largemacromolecules or agents such as proteins, genetic material orattenuated viruses that may easily be degraded or inactivated. Moreover,selected embodiments of the present invention further serve to maintainthe biological activity of incorporated agents by forming relativelystable dispersions comprising nonaqueous suspension media. Thesedispersions of active powder in suspension medium (preferably a liquidfluorochemical or fluorochemical propellant) tend to be bothbacteriostatic and anhydrous, thereby inhibiting hydrolysis orproteolytic decay of the incorporated agent. It will be appreciated thatsuch compositions have been found to maintain comparatively high levelsof activity over prolonged storage periods. Finally, it has been foundsurprisingly that both the composition of the disclosed powders anddelivery techniques thereof can be adjusted to potentiate or enhance theactivity of the associated bioactive agent. Taken together, theseadvantages of the present invention provide for the efficient deliveryand efficacy of highly active agents to the selected physiological site.

As indicated above the compositions, methods and systems of the instantinvention are useful for the delivery of bioactive agents such aspeptides, polypeptides, bacterial carbohydrates, viruses and geneticmaterial. In this respect, the disclosed invention is particularlyuseful for the administration of vaccines for active immunization (e.g.mucosal and systemic vaccination), immunoglobulins for passiveimmunization, immunomodulators for the treatment of autoimmune diseases,active peptides or proteins, and effectors and expression vectors forgene therapy or vaccination. As will be explained in more detail below,powders comprising the selected agent may be formed through a variety ofdifferent means. Preferably, the powders or microparticulates will be inthe form of perforated microstructures and will comprise additionalcomponents to enhance the stability and/or efficacy of the incorporatedbioactive agent. Optionally, the powders may be formulated in asuspension medium to provide stabilized dispersions.

Particularly preferred classes of bioactive agents will be discussed inmore detail immediately below.

B(i). Antigens and Vaccines (for Active Immunization)

In accordance with the teachings herein, particularly preferredbioactive agents will comprise vaccines. As discussed throughout theinstant specification and accompanying examples, compatible vaccines maycomprise inactivated or killed microbes (e.g. viruses), live attenuatedmicrobes, phages, subunit vaccines such as proteins, peptides orcarbohydrates (e.g. bacterial carbohydrates), genetic material includingreplicons, viral vectors, and plasmids and recombinant molecules such asfusion proteins or chimeric antibodies. Regardless of which type ofagent or biologic is selected, the resulting powdered compositions maybe used to immunize a subject against one or more target antigens.Further, the adjuvant effect or enhanced immunity associated with thedisclosed invention provides for particularly effective immunization.

As defined herein a “target antigen” refers to an antigen, typically aportion of a protein or a peptide, toward which it is desirable toinduce an immune response. Such an antigen may be comprised in apathogen, such as a viral, bacterial, protozoan, fungal, yeast, orparasitic antigen, or may be comprised in a cell, such as a cancer cell.Tumor antigens and viral antigens are especially preferred targetantigens. In the case of genetic vaccines, one or more target antigenswill be expressed by the host following transfection or transformationof autologous cells with the administered genetic material. Conversely,in protein or peptide based vaccines, including those comprisingchimeric or fusion proteins or killed or attenuated microbes, the targetantigen or antigens will be presented directly to the immune system. Ineither case, presentation of the target antigens using the powders ordispersions of the instant invention will provoke the desired immuneresponse. Interestingly, it has been found that when live viruses, orcombinations of live and killed viruses have been used as vaccines inaccordance with the teachings herein, a particularly vigorous immuneresponse is generated by the subject.

Those skilled in the art will appreciate that, in general, an effectiveanti-viral immune response comprises both a cell-mediated response,typically involving Th1/CTL cell response and a B cell-mediated humoralresponse. Whereas a purified protein or killed microbe usually elicit B,Th2 without CTL responses, certain formulations of subunit or killedvaccines, as well as live vaccines can elicit B, Th1 associated with CTLresponses. Preferably, the vaccine compositions of the present inventionwill induce a broad range of immune responses upon administration,including B, Th1 and CTL responses. However, infection with live virusduring vaccination can lead to unacceptable side effects. Therefore thegoal of a successful vaccination strategy is to engage both the cellularand humoral branches of immunity without incurring undue adverseeffects. As will be disclosed below, the compositions of the presentinvention may be used to induce both types of response uponadministration.

In this respect, compatible vaccines may include any molecule, organismor compound that results in the generation of B cell response, a T cellresponse or a combination thereof to the target antigen. As such, theagent actually presented to the host immune system (whether directly orfollowing transformation of host cells) may be an analog, homologue orderivative of the naturally occurring target antigen or moleculecomprising the target antigen. Moreover, the immunization may be localor systemic in nature depending on the type of target antigen presentedand the form of delivery. For example, in particularly preferredembodiments the immunogenic response will be largely mucosal in nature(e.g. within the mucosa-associated lymphoid tissue [MALT] lymphoidsystem). As previously discussed, when foreign antigen is presented bylocal dendritic cells, there is a local amplification and maturation ofT-cells and B-cells, which produce IgA and IgM antibodies in addition tothe IgG antibodies typically induced by systemic delivery of antigen.Such localized immunization, particularly in the nasal passages andsinuses, has been found to be particularly effective in preventinginfection by airborne pathogens such as influenza virus and respiratorysyncytial virus. More generally, the vaccine compositions of the presentinvention may comprise one or more target antigens from a number ofpathogens. For example, but not by way of limitation, the target antigenmay be comprised in an influenza virus, a cytomegalovirus, a herpesvirus (including HSV-I and HSV-II), a vaccinia virus, a hepatitis virus(including but not limited to hepatitis A, B, C, or D), a varicellavirus, a rotavirus, a papilloma virus, a measles virus, an Epstein Barrvirus, a coxsackie virus, a polio virus, an enterovirus, an adenovirus,a retrovirus (including, but not limited to, HIV1 or HIV2), arespiratory syncytial virus, a rubella virus, a Streptococcus bacterium(such as Streptococcus pneumoniae), a Staphylococcus bacterium (such asStaphylococcus aureus), a Hemophilus bacterium (such as Hemophilusunfluenzae), a Listeria bacterium (such as Listeria monocytogenes), aKiebsiella bacterium, a Gram-negative bacillus bacterium, an Escherichiabacterium (such as Escherichia coli), a Salmonella bacterium (such asSalmonella typhimurium), a Vibrio bacterium (such as Vibrio cholerae), aYersinia bacterium (such as Yersinia pestis or Yersiniaenterocoliticus), an Enterococcus bacterium, a Neisseria bacterium (suchas Neisseria meningitidis), a Corynebacterium bacterium (such asCorynebacterium diphtheriae), a Clostridium bacterium (such asClostridium tetani), a Mycoplasma (such as Mycoplasma tuberculosis), aCandida yeast, an Aspergillus fungus, a Mucor fungus, a toxoplasma, anamoeba, a malarial parasite, a trypanosomal parasite, a leishmanialparasite, a helminth, etc. Specific nonlimiting examples of such targetantigens include hemagglutinin, nucleoprotein, M protein, F protein, HBSprotein, gp120 protein of HIV, nef protein of HIV, and listeriolysine.

Regardless of what type of antigen is selected to be the target antigenit will comprise at least one relevant epitope. The term “relevantepitope”, as used herein, refers to an epitope comprised in the targetantigen which is accessible to the immune system. For example, arelevant epitope may be processed after penetration of a microbe into acell or recognized by antibodies on the surface of the microbe ormicrobial proteins. Preferably, an immune response directed toward theepitope confers a beneficial effect; for example, where the targetantigen is a viral protein, an immune response toward a relevant epitopeof the target antigen at least partially neutralizes the infectivity orpathogenicity of the virus. Those skilled in the art will appreciatethat the relevant epitopes may be B-cell or T-cell epitopes.

The term “B cell epitope”, as used herein, refers to a peptide,including a peptide sequence contained within a larger protein, whichcan elicit antibody production by B cells.

For example, and not by way of limitation, the hypervariable region 3loop (“V3 loop”) of the envelope protein of human immunodeficiency virus(“HIV”) type 1 is known to be a B cell epitope. Other examples of knownB cell epitopes which may be used according to the invention, include,but are not limited to, epitopes associated with influenza virusstrains, such as site B of influenza HA 1 hemagglutinin, which has beenshown to be an immunodominant B cell epitope (Li et al., 1992, J. Virol.66:399-404); an epitope of F protein of measles virus (residues 404-414,Parlidos et al., 1992, Eur. J. Immunol. 22:2675-2680); an epitope ofhepatitis virus pre-S1 region, from residues 132-145 (Leclerc, 1991, J.Immunol. 147:3545-3552); and an epitope of foot and mouth disease VP1protein, (residues 141-160, Clarke et al., 1987, Nature 330381-384).Still further B cell epitopes which may be used are known or may beidentified by methods known in the art, as set forth in Caton et al.,1982, Cell 31:417-427.

In additional embodiments of the invention, the peptides may comprise Tcell epitopes. The term “T cell epitope”, as used herein, refers to apeptide, including a peptide sequence within a larger protein, whichwhen associated with MHC self antigens and recognized by a T cell,functionally activates the T cell. In this regard the present inventionprovides for the T_(h) epitopes which, in the context of MHC class IIself antigens, may be recognized by a helper T cell and thereby promotethe facilitation of B cell antibody production via the T_(h) cell.

For example, and not by way limitation, influenza A hemagglutinin (HA)protein of PR8 strain, bears, at amino acid residues 110-120, a T_(h)epitope. Other examples of known T cell epitopes include, but are notlimited to, two promiscuous epitopes of tetanus toxoid (Ho et al., 1990,Eur J. Immunol. 20:477-483); an epitope of cytochrome c, (residues88-103); an epitope of Mycrobacteria heatshock protein, (residues350-369, Vordermir et al., Eur. J. Immunol. 24:2061-2067); an epitope ofhen egg white lysozyme, (residues 48-61, Neilsonet al., 1992, Proc.Natl. Acad. Sci. U.S.A. 89:7380-7383); an epitope of Streptococcus A Mprotein, (residues 308-319, Rossiter et al., 1994, Eur. J. Immunol.24:1244-1247); and an epitope of Staphylococcus nuclease protein,(residues 81-100, de Magistris, 1992, Dell 68:1-20). Still further T_(h)epitopes which may be used in conjunction with the instant invention areknown or may be readily identified by methods known in the art.

As a further example, a relevant epitope may be a CTL epitope, which, inthe context of MHC class I self antigens, may be recognized by acytotoxic T cell and thereby promote CTL-mediated lysis of cellscomprising the target antigen. Nonlimiting examples of such epitopesinclude epitopes of influenza virus nucleoproteins corresponding toamino acid residues 147-161 and 365-379, respectively (Taylor et al.,1989 Immunogenetics 26:267; Townsend et al., 1983, Nature 348:674); LSMVpeptide, (amino acid residues 33-41; Zinkernagel et al., 1974, Nature248:701-702); and ovalbumin peptide, corresponding to amino acidresidues 257-264 (Cerbone et al., 1983, J. Exp. Med 163:603-612).

With regard to genetic vaccines, one or more target antigens will beexpressed by the host following transfection or transformation ofautologous cells with the administered genetic material. The expressedantigen(s) then elicit the desired immune response in the subject. Thoseskilled in the art will appreciate that genetic material may beassociated with the powder in the form of naked molecules (e.g. DNA orRNA) or in a viral vector form. In either case, nucleic acids compatiblewith the invention will preferably encode one or more relevant epitopes,and may optionally further comprise elements that regulate theexpression and/or stability and/or immunogenicity of the relevantepitope.

For example, elements that regulate the expression of the epitopeencoded within the genetic construct include, but are not limited to, apromoter/enhancer element, a transcriptional initiation site, apolyadenylation site, a transcriptional termination site, a ribosomebinding site, a translational start codon, a translational stop codon, asignal peptide, etc. Specific examples include, but are not limited to,a promoter and intron A sequence of the initial early gene ofcytomegalovirus (CMV or SV40 virus (“SV40”); Montgomery et al., 1993,DNA and Cell Biology 12:777-783). Alternatively, more than one epitopemay be expressed within the same open reading frame. Examples of geneticvaccines which may be used according to the invention, and methods fortheir production, are set forth in International Application PublicationNo. WO 94/21797, by Merck & Co. and Vical, Inc., InternationalApplication Publication No. WO 97/21687, by Mt. Sinai, U.S. Pat. Nos.5,589,466 and 5,580,859 and in International Application Publication No.WO 90/11092, by Vical, Inc., the contents of which are incorporated byreference in their entireties.

To provide enhanced stability and/or immunogenicity of the relevantepitope, it may be desirable to present the epitope in the context of alarger peptide or protein. For example, the relevant epitope may beexpressed in the variable region of a chimeric antibody or as a portionof a fusion protein. In other preferred embodiments, it may beadvantageous to administer a full-length protein (e.g. a viral coatprotein) comprising one or more relevant epitopes. Alternatively, it maybe desirable to administer powders or perforated microstructurescomprising combinations or cocktails of immunogenic peptides orproteins. In this regard it will be appreciated that the relevantepitopes may be derived from the same or different pathogens. Withrespect to the latter, opportunistic pathogens may be targeted alongwith the primary disease causing agent. In addition to the broad targetrange, the disclosed compositions may comprise various epitopecombinations. For example, the compositions of the present invention maycomprise nucleic acids or peptides or proteins comprising mixtures of Bcell epitopes, mixtures of T cell epitopes, or combinations of B and Tcell epitopes.

More particularly, the administration of compositions that comprise orexpress more than one relevant epitope may exhibit an unexpectedsynergistic effect. It will be appreciated that such combinationvaccines may prove to be much more efficient at conferring the desiredimmunity with respect to the selected pathogen(s) than compositionscomprising a single nucleic acid species encoding a single relevantepitope. Those skilled in the art will further appreciate that suchsynergism could allow for an effective immunoprophylactic orimmunotherapeutic response to be generated with lower dosing and lessfrequent administration than single-epitope vaccines. Moreover, the useof such multiepitope vaccine compositions may provide more comprehensiveprotection as the induced multi-site immunity would tend to be moreresistant to natural phenotypic variation within a species or rapidmutation of a target antigen by the selected pathogen. Of course,effective immunity may also be imparted by vaccines encoding a single Bor T cell epitope and such compositions are clearly contemplated asbeing within the scope of the present invention.

In addition to the antigens themselves, the current invention permitsmanipulation of the excipient components of the particle shell itself toenhance or modify the immunogenicity of the formulated antigen. Forexample, efficient antigen capture by dendritic cells has been shown tobe facilitated when the antigen uptake is facilitated by the mannosereceptor and hence improves targeting to the lysosomal compartment(Salusto et al, J. Expt Med. 182:389-400, 1995). Therefore,incorporation of a low percent of mannans, or other polysaccharides thatbind to receptors on cells, into the particulates would be predicted toenhance the immunogenicity. Furthermore, as will be discussed in moredetail below, the use of cofactor or cytokines to promote APC responsesmight also serve to enhance or suppress an immune response as required.The current invention permits for co-formulation of antigen withcofactors that might augment stimulation local immune responses withinthe mucosa or other targeted sites of delivery (e.g. transdermal)directed to local dendritic cell or other APC. By facilitating APCactivation and enhancing antigen uptake and presentation within a localenvironment, such combination formulations provided by the currentinvention could lead to increased efficiency of the resultant immuneresponse.

More generally, the methods and compositions of the present inventionprovide for an enhanced immune response when used to immunize orvaccinate a subject. This “adjuvant effect” provided by the disclosedparticulates may be used to elicit an immune response comparable to thatelicited by an antigen administered with an adjuvant (i.e. alum orcomplete Freund's adjuvant). Unlike the present invention, those skilledin the art will appreciate that such traditional adjuvants are typicallyassociated with undesirable side effects and, in many cases, are notavailable for use in humans. Conversely, the present invention canafford an enhanced immune response (i.e. an immune response greater thanthat generated by a comparable antigen presented using art recognizedtechniques such as CTL levels for antibody titers), without theadministration of potentially toxic adjuvants. While not wishing to bebound by any particular theory, it is believed that the observedenhancement is, at least in part, a result of the particulateconfiguration or morphology, antigen release profile and possibleantigen aggregation within the particulate. In any event, the effectallows the generation of a clinically useful immune response with lowerlevels of antigen and/or fewer inoculations.

By this adjuvant effect, the immune response provided by thecompositions and methods of the instant invention is enhanced relativeto prior art inoculation techniques. In particular, the immune responseelicited by the compositions of present invention will generally begreater than the immune response provoked by intravenous orintraperitoneal administration of the same antigen solubilized orsuspended in an aqueous carrier. Of course, the magnitude of theelicited immune response may be measured using any one of a variety oftechniques well known to those in the art including compatible methodsset forth in the Examples below. Using such comparisons, thepreparations of the present invention will preferably provoke an immuneresponse that is 25%, 50%, 75% or 100% greater than that provoked byadministration of the same antigen using the prior art methods discussedabove. More preferably, the present invention will provoke a responsethat is 2, 3, 4 or 5 times greater than the baseline response obtainedusing the antigen in an aqueous carrier. In even more preferredembodiments, the disclosed preparations and methods will elicit animmune response that is 6, 7, 8, 9 or even 10 times greater than thebaseline response. Still other embodiments may produce responses thatare 20, 30, 40, 50 times or even two orders of magnitude greater thanbaseline. Those skilled in the art will appreciate that these novel, andheretofore unexpected properties, of the disclosed particulates makethem extremely effective in generating the desired immune response in asubject.

Besides the aforementioned adjuvant effect, other mechanisms may alsocontribute to an enhanced immune response in accordance with theteachings herein. For example, it has surprisingly been found thatcombinations of live and killed virus provoke a much stronger responsethan that provided by the killed virus alone. More particularly, inpreferred embodiments the powders may be formulated using a liveattenuated virus which is, to some extent, killed or inactivated duringthe particulate fabrication. As will be demonstrated in conjunction withthe Examples below, this mixture of live and killed virus appears toelicit a surprisingly strong, or enhanced, immune response. Moreover, inkeeping with the teachings herein, the selected virus or virus mixturemay comprise a naturally occurring inactivated or attenuated virus ormay be engineered to express one or more foreign antigens. Analternative method of formulating live virus provided for by the presentinvention involves the formulation of viral receptors within theparticle matrix followed by binding the selected virus to the particlesafter fabrication (i.e. after spray drying). There are a wide variety ofcellular viral receptors that have now been well defined, for example,the prolactin receptor which can function as a retrovirus receptor,CCR5, the cellular receptor for HIV, the Polio virus receptor, the IgGFc region which binds HSV1 and receptors that bind influenza virus.

Regardless of the antigen selected or the form of the antigen (virus,peptide, genetic material, etc.), those skilled in the art will furtherappreciate that effective immunization of a subject may include morethan one inoculation. As used herein, the terms “immunize” or“immunization” or related terms refer herein to conferring the abilityto mount a substantial immune response (consisting of antibodies orcellular immunity such as effector CTL) against a target antigen orepitope. These terms do not require that completely protective immunitybe created, but rather that a protective immune response be producedwhich is substantially greater than baseline. For example, a mammalianmay be considered to be immunized against a target antigen if thecellular and/or humoral immune response to the target antigen isenhanced following the application of methods of the invention. Assaysdemonstrating the enhancement of both B cell or T cell responses arewell known and could easily be performed by those skilled in the art.Preferably, immunization results in significant resistance to thedisease caused or triggered by pathogens expressing target antigens.

Similarly, the term “inoculating”, as used herein, refers toadministering or introducing a composition comprising at least onevaccine comprising a relevant epitope, or capable of generating orexpressing a relevant epitope, according to the instant disclosure.While an effective immune response may be induced with a singleinoculation, effective immunization of a subject may comprise multipleinoculations or a subsequent booster or boosters. As such, the methodsof the present invention may comprise one, two, three, four or even fiveinoculations in order to achieve the desired immunoprophylactic effect.Moreover, as previously alluded to the administered vaccine willpreferably contact and/or be absorbed by a mucosal surface. Inparticularly preferred embodiments, the mucosal surface will beassociated with oral or nasal passages or cavities or a pulmonary airpassage. Those skilled in the art will further appreciate that thevaccine compositions of the present invention (i.e. powders ordispersions) may be used to inoculate neonates (0-6 mo), infants (6 mo-2yr), children (2 yr-13 yr) or adults (13 yr+).

B(ii). Immunoglobulins (Passive Immunotherapy)

While the methods and compositions of the present invention provideeffective means for inducing localized and systemic active immunity,they may also be used for the induction of localized or systemic passiveimmunity. In particular, the disclosed powders and microparticulates maybe used to administer immunoglobulins, or fragments or portions thereof,to provide rapid prophylaxis or therapy with regard to infection ordisease. The administered immunoglobulins, which may be monoclonal orpolyclonal, will recognize at least one antigen on the target pathogen.Preferably, the recognized antigen or antigens will comprise one or morerelatively conserved epitopes. For the purposes of the presentinvention, the administered compositions may comprise neutralizing,therapeutic or prophylactic antibodies or combinations thereof. Inparticularly preferred embodiments, the administered compositions willcomprise one or more species of monoclonal antibodies or immunoreactivefragments.

Following administration, the active immunoglobulin or immunoglobulinscan either function at the site of delivery or be taken up into thesystemic circulation. Antibodies retained at the site of administrationcould rapidly bind to any target infectious agent (e.g. an airbornevirus) coming in contact with the treated site (i.e. a mucosal surface)and prevent subsequent infection or clear the microbes. Alternatively,the relatively high levels of circulating antibodies provided bypreferred embodiments of the instant invention will allow rapidclearance of the target pathogen from the bloodstream therebypreventing, or at least ameliorating, symptoms associated withinfection. Of course it will be appreciated that, unlike activeimmunization which can last for the lifetime of the subject, passiveimmunization is relatively transitory, lasting as long as the deliveredimmunoglobulin dose remains in the circulation.

As alluded to above any immunoglobulin, or immunoreactive fragmentthereof, that recognizes an antigen or antigens on a target pathogen,may be used to confer the desired immunity on a subject. The ability toprovide both monoclonal and polyclonal antibodies to particularpathogens and/or antigens and/or epitopes is well known in the art. Withrespect to the form of antibody actually administered, it will beappreciated that both native and engineered antibodies are compatiblewith the teachings herein, as are different classes of antibodiesincluding IgA, IgD, IgE, IgG and IgM. Similarly, any immunoreactivefragment or domain of an immunoglobulin, including F(ab′)₂, Fab, or Fvcan be used to provide the desired protection. Regarding engineeredantibodies, humanized constructs (i.e. chimeric antibodies) areparticularly preferred. While such immunoglobulins typically contain theantigen binding complementarity determining regions (CDRs) of murineantibodies, the remainder of the molecule is comprises human antibodysequences which are not recognized as foreign. See, for example, Joneset al., Nature, 321:522-525 (1986) which is incorporated herein byreference. As human polyclonal IgG is not typically recognized asforeign by the subject, these antibodies do not tend to produceundesirable side effects if infrequently administered and are notrapidly eliminated by the body.

Passive immunity is particularly effective in preventing or reducing thechances of infection by readily transmitted pathogens, particularlythose that are air or water borne. As such, powders and dispersions ofthe present invention comprising the appropriate immunoglobulins areespecially effective against respiratory viruses and pathogens such asinfluenza or respiratory syncytial virus. For example, a stabilizeddispersion comprising immunoglobulin laden perforated microstructures ina liquid fluorocarbon medium could be administered to the nasal passagesvia an atomizer or spray bottle. The composition, which could easily beadministered as needed, would provide both localized and systemicpassive immunity with respect to a target pathogen such as a cold virus(Orthomyxovirus, Paramyxovirus, Rhinovirus). Similarly, readilyadministered compositions could be provided in accordance with thepresent invention to provide protection against water borne agents suchas Vibrio cholerae. Passive immunity as disclosed herein could also beused to provide at least some protection with respect to variousorganisms including, but not limited to, Rabies virus, Hepatitis (A, B,C) viruses, HIV and Clostridium tetanii. Other infectious agents forwhich passive immunity may be imparted by the disclosed compositions mayeasily be identified by those skilled in the art.

B(iii). Tumor Antigens

In alternative embodiments, the target antigen may be a tumor antigen.Those skilled in the art will appreciate that tumor antigens are oftenpeptide fragments derived from cell proteins that either are restrictedto the type of tissue from which the tumor is derived or are mutatedduring the course of malignant transformation. Other tumor antigens areoften aberrantly expressed by the tumor cell and/or represent “neo”antigens resulting from errors in transcription, RNA processing due tomutations that are idiosyncratic to the tumor cells. Alternatively,changes in post-translational modifications of a normal protein (e.g.glycosylation) may aid in revealing hitherto hidden (cryptic) epitopesnot normally recognized by the immune system (e.g., as is the case withthe mucin, MUC1). B cell epitopes associated with tumor antigens areexpressed at the surface of tumor cells and are recognized by specificantibodies. In contrast, T cell epitopes are of two types: CTL epitopesthat are MHC class I-restricted peptides derived from tumor associatedantigens and Th epitopes that are MHC class II-restricted peptidesderived from tumor antigens. Whereas Th epitopes are mostly presented byantigen presenting cells (APC) to CD4⁺ T cells, CTL epitopes arepresented by APC as well as tumor cells and are recognized bytumor-specific CD8⁺ T cells. Exemplary tumor antigens include, but arenot limited to, carcinoembryonic antigen (“CEA”), melanoma associatedantigens, alpha fetoprotein, papilloma virus antigens, Epstein Barrantigens, MUC 1, p53, etc. Several other tumor antigens are reportedlyrecognized by autologous cytotoxic T lymphocytes as set forth in Boon,T., et al. J. Exp. Med., 183:725-729, 1996; Disis, M. L., et al. Curr.Opin. Immunol. 8:637-642, 1996; Robbins, P. F., et al. Curr. Opin.Immunol. 8:628-636, 1996, Salgaller et al., J. Surg. Oncol. 68:122-138,1998, each of which is incorporated herein their entirety.

B(iv). Immune Modulation

Autoimmune diseases are mediated by autoreactive T and B cells as wellas other immune cell subtypes that may exert regulatory or effectorroles. It is thought that T cells recognizing organ-specific selfepitopes are a key element in the pathogenesis of autoimmune diseasessuch as diabetes type 1, multiple sclerosis (MS) or rheumatoid arthritis(RA). CD4+ and in certain cases, CD8+T cells recognizing antigenspresented in certain locations of the body may infiltrate the tissue andtrigger destruction of various cell types and persisting inflammation.Whereas Cf4+ Th1 cells that produce IL-2, IFN-, TNF- and LT- areconsidered pathogenic, CD4+ Th2 cells that produce IL-4, IL10, IL-5,IL-13 and IL-9 are considered non-pathogenic relative to autoimmunityand in certain circumstances may suppress disease. Furthermore, Th3cells induced by mucosal exposure to antigens, to secrete TGF- andIL-10, are thought to be crucial mediators of mucosal-induced tolerance.

As a strategy to prevent or suppress the autoimmune diseases,autoreactive T cells provide a good therapeutic target. There areseveral means of inactivating the pathogenic autoreactive T cells(general designation of “tolerance”, which is not necessarily restrictedto “deletion”), responsible for the autoimmune disease: (1) to directlyturn-off or anergize the pathogenic cells by providing long-timeexposure to high levels of antigen; (2) to anergize or switch thefunction of pathogenic T cells by exposing them to antigens in contextof non-professional APC or certain modulating factors; and (3) to induceantigen-specific Th suppressor cells of Th2/Th3 phenotype that migrateto the site of disease and inhibits the function of pathogenic T cells.

Surprisingly, it has been found that tolerance may be induced inaccordance with the present invention through the use of inhalationtherapies. The advantage of the respiratory tract as the target site forimmune tolerance induction is two-fold: first, it is a non-invasiveroute that allows local and systemic delivery of complex antigens; andsecondly, mucosal immunity is likely to comprise Th2/Th3 suppressorcells to the administered antigens. Such antigens may be whole selfantigens (recombinant or purified), antigen fragments (obtained bymolecular biology or biochemical techniques well known in the art) orpeptides limited to epitopes. In other embodiments they may beincorporated as virus components, phages, chimeric antibodies, fusionproteins, replicons, bacteria or delivered via nucleic acid-based orviral vectors. They may be incorporated in self molecules likeimmunoglobulins or any natural or synthetic ligand for receptors on bodycells. They may be administered as isolated, individual components or inmixtures. Examples for diabetes type I include but are not limited tosuch peptides and antigens as: GAD65 (glutamic acid decarboxylase65—Baekkeskov et al., Nature 1990, 347:151), insulin (Palmer et al.,Science 1983, 222:1337), ICA512/IA-2 (islet cell antigen 512; Rabin etal., J. Immunol. 1994, 152:3183). In the case of MS, such proteins andpeptides are: MBP (myelin basic protein, Steinman et al., 1995, Mol.Med. Today, 1:79; Warren et al., 1995, Proc. Natl. Acad. Sci. USA,92:11061). PLP, transaldolase, 2′,3′ cyclic nucleotide 3′phosphodiesterases (CNP), MOG and MAG (Steinman L., 1995, Nature,375:379). Besides autoimmune diseases, it will be appreciated that thecompositions and methods of the present invention may also be used todown regulate immune responses provoked by allergens.

B(v). Active Peptides and Proteins

Certain peptides and proteins are known to have to ability to modulate,up-regulate or down-regulate immune responses to foreign or selfantigens. Such peptides or proteins may act by engaging endogenousreceptors leading to activation or inhibition of certain processes, orby interfering with the ligand-receptor binding of endogenous elements.Examples of such proteins or peptides are cytokines that exert immunemodulatory function leading to suppression of autoimmunity: interferon-,IL4, IL-10, IL-13, IL-9, native or in the form of fragments attached,incorporated or complexed with other molecules. Other cytokines may actas immune activators, leading to increased immunity against microbes ortumor cells: IL-12, IL-2, interferon-, interferon-, TNF-, TNF-,lymphotoxins, and GM-CSF. For example, co-administration of GC-MSF,IFN-α, IL-2, IL-12 or TNF-α has been demonstrated to enhance an immuneresponse and antigen presentation. However, systemic delivery of suchagents in many cases has led to unacceptable side effects, leading to aconcerted effort directed at targeted delivery of these pluripotentfactors. The current invention advantageously permits for co-formulationof a selected antigen or antigens with cofactors that might augmentstimulation local immune responses within the mucosa or other targetedsites of delivery (e.g. transdermal or intradermal) directed to localdendritic cell or other APC presentation. By facilitating APC activationand enhancing antigen uptake and presentation within a local environmentsuch combination formulations of the current invention could provide forincreased efficiency of the resultant immune response.

Other active proteins or peptides that may be used in accordance withthe present invention comprise chemokines in native form or asfragments, constructs or complexes with other molecules which mayincrease, modulate or inhibit the recruitment of lymphocytes. Forexample, whereas eotaxin-1, eotaxin-2, TARC, MIP-3b, SLC are thought tomediate the recruitment of Th2 cells, MIG, IP-10, MIP-1, MIP-1 andRANTES are thought to mediate the recruitment of Th1 cells (Sallusto etal., 1998, J. Exp. Med., 187:875; Ward et al., 1998, Immunity, 9:1).Similarly, cytokine or chemokine receptors in native form, or asfragments, recombinant constructs or complexes with other molecules mayinhibit the recruitment or activation of certain lymphocytes. Examplesof cytokine and chemokine receptors that are likely to inhibit ongoingTh1 responses comprise the IL-12 receptor, IFN-receptor, IL-2 receptor,TNF-receptor, CXCR3 or CCR5. Examples of cytokine and chemokinereceptors that are likely to inhibit ongoing Th2 responses are the IL4receptor, IL-13 receptor, IL-9 receptor, IL-10 receptor, CCR3, CCR4 orCCR7. Of course, it will be appreciated that compatible compounds arenot limited to cytokines, chemokines or their receptors, but may includeother ligands or receptors (in native form, fragments, constructs orcomplexes with other molecules) like integrins and homing receptors. Inpreferred embodiments all these categories of compounds may beformulated and administered either locally or systemically via therespiratory tract in order to enhance, suppress, or modulate an immuneresponse.

It will further be appreciated that the perforated microstructuresaccording to the invention may, if desired, contain a combination of twoor more active ingredients. The agents may be provided in combination ina single species of perforated microstructure or individually inseparate species of perforated microstructures. For example, two or moreactive or bioactive agents may be incorporated in a single feed stockpreparation and spray dried to provide a single microstructure speciescomprising a plurality of bioactive agents. Conversely, the individualagents could be added to separate stocks and spray dried separately toprovide a plurality of microstructure species with differentcompositions. These individual species could be added to the suspensionmedium or dry powder dispensing compartment in any desired proportionand placed in the aerosol delivery system as described below.

Based on the foregoing, it will be appreciated by those skilled in theart that a wide variety of bioactive agents may be incorporated in thedisclosed powders. Accordingly, the list of preferred bioactive agentsabove is exemplary only and not intended to be limiting. It will also beappreciated by those skilled in the art that the proper amount ofbioactive agent and the timing of the dosages may be determined for theformulations in accordance with already existing information and withoutundue experimentation.

C. Powder Composition

As may be seen from the discussion above, the present invention may beused to effectively deliver a wide variety of bioactive agents. Whilethe particulates may be formed exclusively by the bioactive agent, theywill preferably comprise one or more additional materials which, inselected embodiments, may comprise absorption enhancers, potentiators,excipients or structural components. More generally, the particulates(i.e. the structural matrix) may be formed of or comprise any materialwhich possesses physical and chemical characteristics that arecompatible with any incorporated active agents. While a wide variety ofmaterials may be used to form the powders, in particularly preferredpharmaceutical embodiments the particulate is associated with, orcomprises, a surfactant such as phospholipid or fluorinated surfactant.Although not required, the incorporation of a compatible surfactant canimprove powder flowability, increase aerosol efficiency, improvedispersion stability, and facilitate preparation of a suspension.Moreover, selected surfactants may also function as absorption enhancersthereby increasing uptake and improving bioactivity of the selectedagent. Of course it will be appreciated that the powders of the presentinvention may also be formed using nothing more than traditionalnon-surfactant excipients and one or more incorporated bioactive agents.

As indicated, the disclosed powders may optionally be associated with,or comprise, one or more surfactants. In accordance with the teachingsherein, these compounds may serve to stabilize any incorporatedbioactive agent, assist in stabilizing particulates suspended in anonaqueous media or potentiate the uptake of an agent at the targetsite. Besides those surfactants associated with the disclosedparticulates, miscible surfactants may optionally be combined in thecase where the microparticles are formulated in a suspension mediumliquid phase. It will be appreciated by those skilled in the art thatthe use of surfactants, while not necessary to practice the instantinvention, may further increase dispersion stability, powderflowability, simplify formulation procedures or increase efficiency ofdelivery. Of course combinations of surfactants, including the use ofone or more in the liquid phase and one or more associated with theperforated microstructures are contemplated as being within the scope ofthe invention. By “associated with or comprise” it is meant that theparticulate or perforated microstructure may incorporate, adsorb,absorb, be coated with or be formed by the surfactant.

In a broad sense, surfactants suitable for use in the present inventioninclude any compound or composition that aids in the formation ofperforated microparticles or provides enhanced suspension stability,improved powder dispersibility or decreased particle aggregation. Thesurfactant may comprise a single compound or any combination ofcompounds, such as in the case of co-surfactants. Particularly preferredsurfactants are nonfluorinated and selected from the group consisting ofsaturated and unsaturated lipids, nonionic detergents, nonionic blockcopolymers, ionic surfactants and combinations thereof. In thoseembodiments comprising stabilized dispersions, such nonfluorinatedsurfactants will preferably be relatively insoluble in the suspensionmedium. It should be emphasized that, in addition to the aforementionedsurfactants, suitable fluorinated surfactants are compatible with theteachings herein and may be used to provide the desired preparations.

Lipids, including phospholipids, from both natural and synthetic sourcesare particularly compatible with the present invention and may be usedin varying concentrations to form the particulates or structural matrix.Generally compatible lipids comprise those that have a gel to liquidcrystal phase transition greater than about 40° C. Preferably theincorporated lipids are relatively long chain (i.e. C₁₆-C₂₂) saturatedlipids and more preferably comprise phospholipids. Exemplaryphospholipids useful in the disclosed stabilized preparations comprise,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,diarachidoylphosphatidylcholine dibehenoylphosphatidylcholine,short-chain phosphatidylcholines, long-chain saturatedphosphatidylethanolamines, long-chain saturated phosphatidylserines,long-chain saturated phosphatidylglycerols, long-chain saturatedphosphatidylinositols, glycolipids, ganglioside GM1, sphingomyelin,phosphatidic acid, cardiolipin; lipids bearing polymer chains such aspolyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone;lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acidssuch as palmitic acid, stearic acid, and oleic acid; cholesterol,cholesterol esters, and cholesterol hemisuccinate.

Compatible nonionic detergents comprise: sorbitan esters includingsorbitan trioleate (Span® 85), sorbitan sesquioleate, sorbitanmonooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitanmonolaurate, and polyoxyethylene (20) sorbitan monooleate, oleylpolyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, laurylpolyoxyethylene (4) ether, glycerol esters, and sucrose esters. Othersuitable nonionic detergents can be easily identified using McCutcheon'sEmulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.) which isincorporated herein in its entirety. Preferred block copolymers includediblock and triblock copolymers of polyoxyethylene and polyoxypropylene,including poloxamer 188 (Pluronic® F-68), poloxamer 407 (Pluronic®F-127), and poloxamer 338. Ionic surfactants such as sodiumsulfosuccinate, and fatty acid soaps may also be utilized. In preferredembodiments the microstructures may comprise oleic acid or its alkalisalt. Due to their excellent biocompatibility characteristics,phospholipids and combinations of phospholipids and poloxamers areparticularly suitable for use in the pharmaceutical embodimentsdisclosed herein.

In addition to the aforementioned surfactants, cationic surfactants orlipids are preferred especially in the case of delivery or RNA or DNA.Examples of suitable cationic lipids include: DOTMA,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTAP,1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB,1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol. Polycationicamino acids such as polylysine, and polyarginine are also contemplated.

Besides those surfactants enumerated above, it will further beappreciated that a wide range of surfactants may optionally be used inconjunction with the present invention. Moreover, the optimum surfactantor combination thereof for a given application can readily be determinedby empirical studies that do not require undue experimentation. Finally,as discussed in more detail below, surfactants comprising theparticulate or structural matrix may also be useful in the formation ofprecursor oil-in-water emulsions (i.e. spray drying feed stock) usedduring processing to form the perforated microstructures.

Unlike prior art formulations, it has surprisingly been found that theincorporation of relatively high levels of surfactants or biocompatiblewall forming material (e.g., phospholipids) may be used to improvepowder dispersibility, increase suspension stability and decrease powderaggregation of the disclosed applications. That is, on a weight toweight basis, the particulate or structural matrix of the perforatedmicrostructures may comprise relatively high levels of surfactant. Inthis regard, the particulates will preferably comprise greater thanabout 1%, 5%, 10%, 15%, 18%, 20% w/w surfactant. More preferably, themicroparticulates or microstructures will comprise greater than about25%, 30%, 35%, 40%, 45%, or 50% w/w surfactant. Still other exemplaryembodiments will comprise particulates wherein the surfactant orsurfactants are present at greater than about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90% or even 95% w/w. In selected embodiments the powders willcomprise essentially 100% w/w of a surfactant such as a phospholipid.Those skilled in the art will appreciate that, in such cases, thebalance of the particulate or structural matrix (where applicable) willlikely comprise a bioactive agent, excipients or other additives.

As will be discussed below, surfactants may be incorporated in any typeof particulate. That is, while the aforementioned surfactant levels arepreferably employed in perforated microstructures, they may be used toprovide powders or stabilized dispersions comprising relativelynonporous, or substantially solid, particulates. While selectedembodiments of the present invention will comprise perforatedmicrostructures associated with high levels of surfactant, compatiblepowders may be formed using relatively low porosity particulates ofequivalent surfactant concentrations. Preferably, such particulates willcomprise relatively high levels of surfactant on the order of greaterthan about 5% w/w. In this respect, such embodiments are specificallycontemplated as being within the scope of the present invention.

In other preferred embodiments of the invention, the particulatesoptionally comprise synthetic or natural polymers or combinationsthereof. In this respect useful polymers comprise polylactides,polylactide-co-glycolides, cyclodextrins, polyacrylates,methylcellulose, carboxymethylcellulose, polyvinyl alcohols,polyanhydrides, polylactams, polyvinyl pyrrolidones, monosaccharides,disaccharides or polysaccharides (dextrans, starches, chitin, chitosan,etc.), hyaluronic acid, proteins, (albumin, collagen, gelatin, etc.).Examples of polymeric resins that might prove useful for the preparationof microparticles include: styrene-butadiene, styreneisoprene,styrene-acrylonitrile, ethylene-vinyl acetate, ethylene-acrylate,ethylene-acrylic acid, ethylene-methylacrylatate, ethylene-ethylacrylate, vinyl-methyl methacrylate, acrylic acid-methyl methacrylate,and vinyl chloride-vinyl, acetate. Those skilled in the art willappreciate that, by selecting the appropriate polymers, the deliveryefficiency of the particulates and/or the stability of the dispersionsmay be tailored to optimize the effectiveness of the active or bioactiveagent.

Besides the aforementioned polymer materials and surfactants, variousexcipients may be incorporated in, or added to, the particulates toprovide structure and, in preferred embodiments form perforatedmicrostructures (i.e. microspheres such as latex particles). In thisregard it will be appreciated that the rigidifying components can beremoved using a post-production technique such as selective solventextraction. Compatible excipients may include, but are not limited to,carbohydrates including monosaccharides, disaccharides andpolysaccharides. For example, monosaccharides such as dextrose(anhydrous and monohydrate), galactose, mannitol, D-mannose, sorbitol,sorbose and the like; disaccharides such as lactose, maltose, sucrose,trehalose, and the like; trisaccharides such as raffinose and the like;and other carbohydrates such as starches (hydroxyethylstarch),cyclodextrins and maltodextrins. Amino acids are also suitableexcipients with glycine preferred. Mixtures of carbohydrates and aminoacids are further held to be within the scope of the present invention.The inclusion of both inorganic (e.g. sodium chloride, calcium chloride,etc.), organic salts (e.g. sodium citrate, sodium ascorbate, magnesiumgluconate, sodium gluconate, tromethamine hydrochloride, etc.) andbuffers is also contemplated. The inclusion of salts and organic solidssuch as ammonium carbonate, ammonium acetate, ammonium chloride orcamphor are also contemplated.

Along with the compounds discussed above, it may be desirable to addother excipients to a microsphere formulation to improve particlerigidity, production yield, delivery efficiency and deposition,shelf-life and patient acceptance. Such optional excipients include, butare not limited to: coloring agents, taste masking agents, buffers,hygroscopic agents, antioxidants, and chemical stabilizers. Moreover, asdiscussed above, the particulates may comprise compounds that canpotentiate, induce or modulate the uptake of the associated bioactiveagent. Further, the particulates of the invention may comprise targetingmolecules such as antibodies, cofactors, receptors, ligands andsubstrates that preferentially direct the particulates, or allow them tobind, to molecules associated with cells at the target site. Forexample, particulates could be formed comprising an antibody targeting amucosal cell receptor and an immunoactive compound. Such targetingmolecules would likely increase the concentration of bioactiveparticulates at the target mucosal site and further enhance anylocalized immune response. It will be appreciated that ligands directedto receptors preferentially expressed on the surface of mucosal or othertarget cells could also be used to increase the binding of particulatesat the desired site.

Yet other preferred embodiments include perforated microstructures thatmay comprise, or may be coated with, charged species that prolongresidence time at the point of contact or enhance penetration throughmucosae. For example, anionic charges are known to favor mucoadhesionwhile cationic charges may be used to associate the formedmicroparticulate with negatively charged bioactive agents such asgenetic material. The charges may be imparted through the association orincorporation of polyanionic or polycationic materials such aspolyacrylic acids, polylysine, polylactic acid and chitosan.

D. Powder Morphology

Those skilled in the art will appreciate that powders or particulates ofvarious compositions, configurations and morphologies may be used inaccordance with the present invention as long as they provide desiredstability and delivery characteristics. In this respect, it may beadvantageous to use relatively dense, solid particulates or powders forsome applications (e.g. for intradermal administration of a stabilizeddispersion via a air gun or needleless injector) while in otherembodiments (e.g. DPI administration) a relatively porous,aerodynamically light perforated microstructure may be preferred.Accordingly, while the present invention may be discussed below in termsof preferred embodiments, it must be emphasized that it is not limitedto any particular particle composition, configuration or morphology.Rather, selection of particulate characteristics (charge, density,composition, etc.) is largely based on the form of administration,targeted delivery site and choice of bioactive agent.

While various particulate configurations, including micronized andmilled particulates, may be used in accordance with the teachingsherein, the present invention provides unique methods and compositionsto reduce cohesive forces between dry particles, thereby minimizingparticulate aggregation that can result in improved delivery efficiency.As such, selected disclosed preparations provide a highly flowable, drypowders that can be efficiently aerosolized, uniformly delivered andpenetrate deeply in the lung or nasal passages. Moreover, selectedpowder configurations and morphologies have been found to providerelatively stable dispersions when combined with a nonaqueous suspensionmedium. In either case, the disclosed particulates may be fabricated soas to result in surprisingly low throat deposition upon administration.

As previously discussed, particularly preferred embodiments of thepresent invention incorporate powders or particulates in the form ofporous or perforated microstructures comprising a structural matrix. Itwill be appreciated that, as used herein, the terms “structural matrix”or “microstructure matrix” are equivalent and shall be held to mean anysolid material forming perforated microstructures which define aplurality of voids, apertures, hollows, defects, pores, holes, fissures,etc. that provide the desired characteristics. In selected embodiments,the perforated microstructures defined by the structural matrix comprisea spray dried hollow porous microsphere incorporating at least onesurfactant. It will further be appreciated that, by altering the matrixcomponents, the density of the structural matrix may be adjusted so asto further increase dispersion stability or delivery efficiency.

The absolute shape (as opposed to the morphology) of the particulates orperforated microstructures is generally not critical and any overallconfiguration that provides the desired characteristics is contemplatedas being within the scope of the invention. Accordingly, preferredembodiments can comprise approximately microspherical shapes. However,collapsed, deformed or fractured particulates are also compatible. Withthis caveat, it will further be appreciated that, particularly preferredembodiments of the invention comprise spray dried hollow, porousmicrospheres. In any case the disclosed powders of perforatedmicrostructures provide several advantages including, but not limitedto, increases in suspension stability, improved dispersibility, superiorsampling characteristics, elimination of carrier particles and enhancedaerodynamics.

To maximize dispersibility, dispersion stability and optimizedistribution upon administration, the mean geometric particle size ofthe particulates or perforated microstructures is preferably about0.5-50 μm, more preferably 1-30 μm. It will be appreciated that largeparticles (i.e. greater than 50 μm) may not be preferred in applicationswhere a valve or small orifice is employed, since large particles tendto aggregate or separate from a suspension which could potentially clogthe device. In especially preferred embodiments the mean geometricparticle size (or diameter) of the perforated microstructures is lessthan 20 μm or less than 1 μm. More preferably the mean geometricdiameter is less than about 7 μm or 5 μm, and even more preferably lessthan about 4 μm or even 2.5 μm. Other preferred embodiments willcomprise preparations wherein the mean geometric diameter of theperforated microstructures is between about 1 μm and 5 μm. In especiallypreferred embodiments the perforated microstructures will comprise apowder of dry, hollow, porous microspherical shells of approximately 1to 10 μm or 1 to 5 μm in diameter, with shell thicknesses ofapproximately 0.1 μm to approximately 0.5 μm. It is a particularadvantage of the present invention that the particulate concentration ofthe dispersions and structural matrix components can be adjusted tooptimize the delivery characteristics of the selected particle size.

As alluded to throughout the instant specification the porosity of themicrostructures may play a significant part is establishingdispersibility (e.g. in DPIs) or dispersion stability (e.g. for MDIs,jet guns or nebulizers). In this respect, the mean porosity of theperforated microstructures may be determined through electron microscopycoupled with modern imaging techniques. More specifically, electronmicrographs of representative samples of the perforated microstructuresmay be obtained and digitally analyzed to quantify the porosity of thepreparation. Such methodology is well known in the art and may beaccomplished without undue experimentation.

For the purposes of the present invention, the mean porosity (i.e. thepercentage of the particle surface area that is open to the interiorand/or a central void) of the particulates or perforated microstructuresmay range from approximately 0.5% to approximately 80%. In morepreferred embodiments, the mean porosity will range from approximately2% to approximately 40%. Based on selected production parameters, themean porosity may be greater than approximately, 2%, 5%, 10%, 15%, 20%,25% or 30% of the microstructure surface area. In other embodiments themean porosity of the microstructures may be greater than about 40%, 50%,60%, 70% or even 80%. As to the pore themselves, they typically range insize from about 5 nm to about 400 nm with mean pore sizes preferably inthe range of from about 20 nm to about 200 nm. In particularly preferredembodiments the mean pore size will be in the range of from about 50 nmto about 100 nm. As will be discussed in more detail below, it is asignificant advantage of the present invention that the pore size andporosity may be closely controlled by careful selection of theincorporated components and production parameters.

In this regard, the particle morphology and/or hollow design of theparticulates or perforated microstructures also plays an important roleon the dispersibility or cohesiveness of the dry powder formulationsdisclosed herein. That is, it has been surprisingly discovered that theinherent cohesive character of fine powders can be overcome by loweringthe van der Waals, electrostatic attractive and liquid bridging forcesthat typically exist between dry particles. More specifically, inconcordance with the teachings herein, improved powder dispersibilitymay be provided by engineering the particle morphology and density, aswell as control of humidity and charge. To that end, preferredembodiments of the present invention comprise perforated microstructureshaving pores, voids, hollows, defects or other interstitial spaces whichreduce the surface contact area between particles thereby minimizinginterparticle forces. In addition, the use of surfactants such asphospholipids and fluorinated blowing agents in accordance with theteachings herein may contribute to improvements in the flow propertiesof the powders by tempering the charge and strength of the electrostaticforces as well as moisture content.

Most fine powders (e.g. <5 μm) exhibit poor dispersibility which can beproblematic when attempting to deliver, aerosolize and/or package thepowders. In this respect the major forces which control particleinteractions can typically be divided into long and short range forces.Long range forces include gravitational attractive forces andelectrostatics, where the interaction varies as a square of theseparation distance or particle diameter. Important short range forcesfor dry powders include van der Waals interactions, hydrogen bonding andliquid bridges. The latter two short range forces differ from the othersin that they occur where there is already contact between particles. Itis a major advantage of the present invention that these attractiveforces may be substantially attenuated or reduced through the use ofperforated microstructures as described herein.

Those skilled in the art will appreciate that the van der Waals (VDW)attractive force occurs at short range and depends, at least in part, onthe surface contact between the interacting particles. When twoparticles approach each other the VDW forces increase with an increasein contact area. For two dry particles, the magnitude of the VDWinteraction force, F⁰ _(vdw), can be calculated using the followingequation:$F_{vdw}^{0} = {\frac{\hslash \quad \omega}{8\quad \pi \quad d_{0}^{2}}\lbrack \frac{r_{1}\quad r_{2}}{r_{1} + r_{2}} \rbrack}$

where  is Planck's constant, ω is the angular frequency, d₀ is thedistance at which the adhesional force is at a maximum, and r₁, and r₂are the radii of the two interacting particles. Accordingly, i will beappreciated that one way to minimize the magnitude and strength of theVDW force for dry powders is to decrease the interparticle area ofcontact. It is important to note that the magnitude of do is areflection of this area of contact. The minimal area of contact betweentwo opposing bodies will occur if the particles are perfect spheres. Inaddition, the area of contact will be further minimized if the particlesare highly porous. Accordingly, the perforated microstructures of thepresent invention act to reduce interparticle contact and correspondingVOW attractive forces. It is important to note that this reduction inVDW forces is largely a result of the unique particle morphology of thepowders of the present invention rather than an increase in geometricparticle diameter. In this regard, it will be appreciated thatparticularly preferred embodiments of the present invention providepowders having average or small particulates (e.g. mean geometricdiameter<10 μm) exhibiting relatively low VDW attractive forces.

Further, as indicated above, the electrostatic force affecting powdersoccurs when either or both of the particles are electrically charged.This phenomenon will result with either an attraction or repulsionbetween particles depending on the similarity or dissimilarity ofcharge. In the simplest case, the electric charges can be describedusing Coulomb's Law. One way to modulate or decrease the electrostaticforces between particles is if either or both particles havenon-conducting surfaces. Thus, if the perforated microstructure powderscomprise excipients, surfactants or active agents that are relativelynonconducting, then any charge generated in the particle will beunevenly distributed over the surface. As a result, the charge half-lifeof powders comprising non-conducting components will be relatively shortsince the retention of elevated charges is dictated by the resistivityof the material. Resistive or non-conducting components are materialswhich will neither function as an efficient electron donor or acceptor.

Derjaguin et al. (Muller, V. M., Yushchenko, V. S., and Derjaguin, B.V., J. Colloid Interface Sci. 1980, 77, 115-119), which is incorporatedherein by reference, provide a list ranking molecular groups for theirability to accept or donate an electron. In this regard exemplary groupsmay be ranked as follows:

Donor:—NH₂>—OH>—OR>—COOR>—CH₃>—C₆H₅>-halogen>—COOH>—CO>—CN Acceptor:

The present invention provides for the reduction of electrostaticeffects in the disclosed powders though the use of relativelynon-conductive materials. Using the above rankings, preferrednon-conductive materials would include halogenated and/or hydrogenatedcomponents. Materials such as phospholipids and fluorinated blowingagents (which may be retained to some extent in spray dried powders) arepreferred since they can provide resistance to particle charging. Itwill be appreciated that the retention of residual blowing agent (e.g.fluorochemicals) in the particles, even at relatively low levels, mayhelp minimize charging of particulates or perforated microstructures asis typically imparted during spray drying and cyclone separation. Basedon general electrostatic principles and the teachings herein, oneskilled in the art would be able to identify additional materials thatserve to reduce the electrostatic forces of the disclosed powderswithout undue experimentation. In this regard, highly charged agents canbe electrostatically modified and controlled through simple pHadjustments or chelation with oppositely charged compounds, e.g.associating nucleic acids with cationic lipids. Further, if needed, theelectrostatic forces can also be manipulated and minimized usingelectrification and charging techniques.

In addition to the surprising advantages described above, the presentinvention further provides for the attenuation or reduction of hydrogenand liquid bonding. As known to those skilled in the art, both hydrogenbonding and liquid bridging can result from moisture that is absorbed bythe powder. In general, higher humidities produce higher interparticleforces for hydrophilic surfaces. This is a substantial problem in priorart pharmaceutical formulations for inhalation therapies which tend toemploy relatively hydrophilic compounds such as lactose. However, inaccordance with the teachings herein, adhesion forces due to adsorbedwater can be modulated or reduced by increasing the hydrophobicity ofthe contacting surfaces. One skilled in the art can appreciate that anincrease in particle hydrophobicity can be achieved through excipientselection and/or use a post-production spray drying coating techniquesuch as employed using a fluidized bed. Thus, preferred excipientsinclude hydrophobic surfactants such as phospholipids, fatty acid soapsand cholesterol. In view of the teachings herein, it is submitted that askilled artisan would be able to identify materials exhibiting similardesirable properties without undue experimentation.

Whether they are to be used as a dry powder or combined with anonaqueous suspension medium, the particulates or perforatedmicrostructures will preferably be provided in a “dry” state. That isthe microparticles will possess a moisture content that allows thepowder to remain chemically and physically stable during storage atambient temperature and easily dispersible. As such, the moisturecontent of the microparticles is typically less than 6% by weight, andpreferably less 3% by weight. In some instances the moisture contentwill be as low as 1% by weight. Of course it will be appreciated thatthe moisture content is, at least in part, dictated by the formulationand is controlled by the process conditions employed, e.g., inlettemperature, feed concentration, pump rate, and blowing agent type,concentration and post drying.

As known by those skilled in the art, methods such as angle of repose orshear index can be used to assess the flow properties of dry powders.The angle of repose is defined as the angle formed when a cone of powderis poured onto a flat surface. Powders having an angle of repose rangingfrom 45° to 20° are preferred and indicate suitable powder flow. Moreparticularly, powders which possess an angle of repose between 33° and20° flow with relatively low shear forces and are especially useful inpharmaceutical preparations for use in inhalation therapies (e.g. DPIs).The shear index, though more time consuming to measure than angle ofrepose, is considered more reliable and easy to determine. Those skilledin the art will appreciate that the experimental procedure outlined byAmidon and Houghton (G. E. Amidon, and M. E. Houghton, Pharm. Manuf., 2,20, 1985, incorporated herein by reference) can be used estimate theshear index for the purposes of the present invention. As described inS. Kocova and N. Pilpel, J. Pharm. Pharmacol. 8, 33-55, 1973, alsoincorporated herein by reference, the shear index is estimated frompowder parameters such as, yield stress, effective angle of internalfriction, tensile strength, and specific cohesion. In the presentinvention powders having a shear index less than about 0.98 aredesirable. More preferably, powders used in the disclosed compositions,methods and systems will have shear indices less than about 1.1. Inparticularly preferred embodiments the shear index will be less thanabout 1.3 or even less than about 1.5. Of course powders havingdifferent shear indices may be used provided the result in the effectivedeposition of the active or bioactive agent at the site of interest.

It will also be appreciated that the flow properties of powders havebeen shown to correlate well with bulk density measurements. In thisregard, conventional prior art thinking (C. F. Harwood, J. Pharm Sci.,60, 161-163, 1971) held that an increase in bulk density correlates withimproved flow properties as predicted by the shear index of thematerial. Conversely, it has surprisingly been found that, for theperforated microstructures of the present invention, superior flowproperties were exhibited by powders having relatively low bulkdensities. That is, the hollow porous powders of the present inventionexhibited superior flow properties over powders substantially devoid ofpores. To that end, it has been found that it is possible to providepowders having bulk densities of less than 0.5 g/cm³ that exhibitparticularly favorable flow properties. More surprisingly, it has beenfound that it is possible to provide perforated microstructure powdershaving bulk densities of less than 0.3 g/cm³, less than about 0.1 g/cm³or even on the order of 0.05 g/cm³that exhibit excellent flowproperties. The ability to produce low bulk density powders havingsuperior flowability further accentuates the novel and unexpected natureof the present invention.

These low bulk densities are particularly advantageous when using thedisclosed powders in conjunction with DPIs. Specifically, by affordingpowder formulations having extraordinarily low bulk density, the presentinvention allows for reduction of the minimal filling weight that iscommercially feasible for use in dry powder inhalation devices. That is,most unit dose containers designed for DPIs are filled using fixedvolume or gravimetric techniques. Contrary to many prior artformulations, the present invention provides powders wherein bioactiveagent and the incipients or bulking agents make-up the entire inhaledparticle. By providing particles with very low bulk density, the minimumpowder mass that can be filled into a unit dose container is reduced,which eliminates the need for carrier particles. That is, the relativelylow density of the powders of the present invention provides for thereproducible administration of relatively low dose pharmaceuticalcompounds without the use of carrier particles. Moreover, theelimination of carrier particles acts to minimize throat deposition andany “gag” effect, since the large lactose particles of prior artformulations tend to impact the throat and upper airways due to theirsize.

It will be appreciated that the reduced attractive forces (e.g. van derWaals, electrostatic, hydrogen and liquid bridging, etc.) and excellentflowability provided by the perforated microstructure powders make themparticularly useful in preparations for inhalation therapies (e.g. ininhalation devices such as DPIs, MDIs, nebulizers). Along with thesuperior flowability, the perforated or porous and/or hollow design ofthe microstructures also plays an important role in the resultingaerosol properties of the powder when discharged. This phenomenon holdstrue for particulates or perforated microstructures aerosolized as asuspension, as in the case of an MDI or a nebulizer, or delivery ofperforated microstructures in dry form as in the case of a DPI. In thisrespect the perforated structure and relatively high surface area of thedispersed microparticles enables them to be carried along in the flow ofgases during inhalation with greater ease for longer distances thannon-perforated particles of comparable size.

More particularly, because of their high porosity, the density of theparticles is significantly less than 1.0 g/cm³, typically less than 0.5g/cm³, more often on the order of 0.1 g/cm³, and as low as 0.01 g/cm³.Unlike the geometric particle size, the aerodynamic particle size,d_(aer), of the perforated microstructures depends substantially on theparticle density, ρ: d_(aer)=d_(geo)ρ, where d_(geo) is the geometricdiameter. For a particle density of 0.1 g/cm³, d_(aer) will be roughlythree times smaller than d_(geo), leading to increased particledeposition into the peripheral regions of the lung and correspondinglyless deposition in the throat. In this regard, the mean aerodynamicdiameter of the perforated microstructures is preferably less than about5 μm, more preferably less than about 3 μm, and, in particularlypreferred embodiments, less than about 2 μm. Such particle distributionswill act to increase the deep lung deposition of the bioactive agentwhether administered using a DPI, MDI or nebulizer.

As will be shown subsequently in the Examples, the particle sizedistribution of the aerosol formulations of the present invention aremeasurable by conventional techniques such as, for example, cascadeimpaction or by time of flight analytical methods. In addition,determination of the emitted dose from inhalation devices were doneaccording to the proposed U.S. Pharmacopeia method (PharmacopeialPreviews, 22(1996) 3065) which is incorporated herein by reference.These and related techniques enable the “fine particle fraction” of theaerosol, which corresponds to those particulates that are likely toeffectively deposited in the lung, to be calculated. As used herein thephrase “fine particle fraction” refers to the percentage of the totalamount of active medicament delivered per actuation from the mouthpieceof a DPI, MDI or nebulizer onto plates 2-7 of an 8 stage Andersencascade impactor. Based on such measurements the formulations of thepresent invention will preferably have a fine particle fraction ofapproximately 20% or more by weight of the perforated microstructures(w/w), more preferably they will exhibit a fine particle fraction offrom about 25% to 90% w/w, and even more preferably from about 30 to 80%w/w. In selected embodiments the present invention will preferablycomprise a fine particle fraction of greater than about 30%, 40%, 50%,60%, 70%, 80% or even 90% by weight.

Further, it has also been found that the formulations of the presentinvention exhibit relatively low deposition rates, when compared withprior art preparations, on the induction port and onto plates 0 and 1 ofthe impactor. Deposition on these components is linked with depositionin the throat in humans. More specifically, most commercially availableMDIs and DPIs have simulated throat depositions of approximately 40-70%(w/w) of the total dose, while the formulations of the present inventiontypically deposit less than about 20% w/w. Accordingly, preferredembodiments of the present invention have simulated throat depositionsof less than about 40%, 35%, 30%, 25%, 20%, 15% or even 10% w/w. Thoseskilled in the art will appreciate that significant decrease in throatdeposition provided by the present invention will result in acorresponding decrease in associated local side-effects such as throatirritation.

With respect to the advantageous deposition profile provided by theinstant invention it is well known that MDI propellants typically forcesuspended particles out of the device at a high velocity towards theback of the throat. Since prior art formulations typically contain asignificant percentage of large particles and/or aggregates, as much astwo-thirds or more of the emitted dose may impact the throat. Moreover,the undesirable delivery profile of conventional powder preparations isalso exhibited under conditions of low particle velocity, as occurs withDPI devices. In general, this problem is inherent when aerosolizingsolid, dense, particulates which are subject to aggregation. Yet, asdiscussed above, the novel and unexpected properties of the stabilizeddispersions of the present invention result in surprisingly low throatdeposition upon administration from inhalation device such as a DPI, MDIatomizer or nebulizer.

While not wishing to be bound by any particular theory, it appears thatthe reduced throat deposition provided by the instant invention resultsfrom decreases in particle aggregation and from the hallow and/or porousmorphology of the incorporated microstructures. That is, the hollow andporous nature of the dispersed microstructures slows the velocity ofparticles in the propellant stream (or gas stream in the case of DPIs),just as a hollow/porous whiffle ball decelerates faster than a baseball.Thus, rather than impacting and sticking to the back of the throat, therelatively slow traveling particles are subject to inhalation by thepatient. Moreover, the highly porous nature of the particles allows thepropellant within the perforated microstructure to rapidly leave and theparticle density to drop before impacting the throat. Accordingly, asubstantially higher percentage of the administered bioactive agent isdeposited in the pulmonary air passages where it may be efficientlyabsorbed.

E. Powder Formation

As seen from the passages above, various components may be associatedwith, or incorporated in the microparticulates of the present invention.Similarly, several techniques may be used to provide particulates havingthe desired morphology (e.g. a perforated or hollow/porousconfiguration), dispersibility and density. Among other methods,particulates compatible with the instant invention may be formed bytechniques including spray drying, vacuum drying, solvent extraction,emulsification, lyophilization and combinations thereof. It will furtherbe appreciated that the basic concepts of many of these techniques arewell known in the prior art and would not, in view of the teachingsherein, require undue experimentation to adapt them so as to provide thedesired particle configuration and/or density.

While several procedures are generally compatible with the presentinvention, particularly preferred embodiments typically compriseparticulates or perforated microstructures formed by spray drying. As iswell known, spray drying is a one-step process that converts a liquidfeed to a dried particulate form. With respect to pharmaceuticalapplications, it will be appreciated that spray drying has been used toprovide powdered material for various administrative routes includinginhalation. See, for example, M. Sacchetti and M. M. Van Oort in:Inhalation Aerosols: Physical and Biological Basis for Therapy, A. J.Hickey, ed. Marcel Dekkar, New York, 1996, which is incorporated hereinby reference.

In general, spray drying consists of bringing together a highlydispersed liquid, and a sufficient volume of hot air to produceevaporation and drying of the liquid droplets. The preparation to bespray dried or feed (or feed stock) can be any solution, coursesuspension, slurry, colloidal dispersion, or paste that may be atomizedusing the selected spray drying apparatus. In preferred embodiments thefeed stock will comprise a colloidal system such as an emulsion, reverseemulsion, microemulsion, multiple emulsion, particulate dispersion, orslurry. Typically the feed is sprayed into a current of warm filteredair that evaporates the solvent and conveys the dried product to acollector. The spent air is then exhausted with the solvent. Thoseskilled in the art will appreciate that several different types ofapparatus may be used to provide the desired product. For example,commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. willeffectively produce particles of desired size, morphology and density.

It will further be appreciated that these spray dryers, and specificallytheir atomizers, may be modified or customized for specializedapplications, i.e. the simultaneous spraying of two solutions using adouble nozzle technique. More specifically, a water-in-oil emulsion canbe atomized from one nozzle and a solution containing an anti-adherentsuch as mannitol can be co-atomized from a second nozzle. In other casesit may be desirable to push the feed solution though a custom designednozzle using a high pressure liquid chromatography (HPLC) pump. Providedthat microstructures comprising the desired morphology and/orcomposition are produced, the choice of apparatus is not critical andwould readily be apparent to the skilled artisan in view of theteachings herein.

While the resulting spray-dried powders typically are approximatelyspherical in shape, nearly uniform in size and frequently are hollow,there may be some degree of irregularity in shape depending upon theincorporated medicament and the spray drying conditions. In manyinstances dispersion stability and dispersibility of particulates orperforated microstructures appears to be improved if an inflating agent(or blowing agent) is used in their production. Particularly preferredembodiments may comprise an emulsion with the inflating agent as thedisperse or continuous phase. The inflating agent is preferablydispersed with a surfactant solution, using, for instance, acommercially available microfluidizer at a pressure of about 5000 to15,000 psi. This process forms an emulsion, preferably stabilized by anincorporated surfactant, typically comprising submicron droplets ofwater immiscible blowing agent dispersed in an aqueous continuous phase.The formation of such emulsions using this and other techniques arecommon and well known to those in the art. The blowing agent ispreferably a fluorinated compound (e.g. perfluorohexane, perfluorooctyl,bromide, perfluorodecalin, perfluorobutyl ethane) which vaporizes duringthe spray-drying process, leaving behind, in selected embodiments,relatively hollow, porous aerodynamically light microspheres. As will bediscussed in more detail below, other suitable liquid blowing agentsinclude nonfluorinated oils, chloroform, Freons, ethyl acetate, alcoholsand hydrocarbons. Nitrogen and carbon dioxide gases are alsocontemplated as suitable blowing agents.

Besides the aforementioned compounds, inorganic and organic substanceswhich can be removed under reduced pressure by sublimation in apost-production step are also compatible with the instant invention.These sublimating compounds can be dissolved or dispersed as micronizedcrystals in the spray drying feed solution and include ammoniumcarbonate and camphor. Other compounds compatible with the presentinvention comprise rigidifying solid structures which can be dispersedin the feed solution or prepared in-situ. These structures are thenextracted after the initial particle generation using a post-productionsolvent extraction step. For example, latex particles can be dispersedand subsequently dried with other wall forming compounds, followed byextraction with a suitable solvent.

Although the particulates are preferably formed using a blowing agent asdescribed above, it will be appreciated that, in some instances, noadditional blowing agent is required and an aqueous dispersion of themedicament and/or excipients and surfactant(s) are spray dried directly.In such cases, the formulation may be amenable to process conditions(e.g., elevated temperatures) that may lead to the formation of hollow,relatively porous microparticles. Moreover, the medicament may possessspecial physicochemical properties (e.g., high crystallinity, elevatedmelting temperature, surface activity, etc.) that makes it particularlysuitable for use in such techniques.

When a blowing agent is employed, the degree of porosity anddispersibility of the resulting particulates appears to depend, at leastin part, on the nature of the blowing agent, its concentration in thefeed stock (e.g. as an emulsion), and the spray drying conditions. Withrespect to controlling porosity and, in suspensions, dispersibility ithas surprisingly been found that the use of compounds, heretoforeunappreciated as blowing agents, may provide perforated microstructureshaving particularly desirable characteristics. More particularly, inthis novel and unexpected aspect of the present invention it has beenfound that the use of fluorinated compounds having relatively highboiling points (i.e. greater than about 40° C.) may be used to produceparticulates that are especially porous. Such perforated microstructuresare especially suitable for inhalation therapies. In this regard it ispossible to use fluorinated or partially fluorinated blowing agentshaving boiling points of greater than about 40° C., 50° C., 60° C., 70°C., 80° C., 90° C. or even 95° C. Particularly preferred blowing agentshave boiling points greater than the boiling point of water, i.e.greater than 100° C.(e.g. perflubron, perfluorodecalin). In additionblowing agents with relatively low water solubility (<10⁻⁶ M) arepreferred since they facilitate the production of stable emulsiondispersions with mean weighted particle diameters less than 0.3 μm.

As previously described, these blowing agents will preferably beincorporated in an emulsified feed stock prior to spray drying. For thepurposes of the present invention this feed stock will also preferablycomprise one or more bioactive agents, one or more surfactants or one ormore excipients. Of course, combinations of the aforementionedcomponents are also within the scope of the invention. While highboiling (>100° C.) fluorinated blowing agents comprise one preferredaspect of the present invention, it will be appreciated thatnonfluorinated blowing agents with similar boiling points (>100° C.) maybe also be used to provide compatible particulates. Exemplarynonfluorinated blowing agents suitable for use in the present inventioncomprise the formula:

R¹—X—R² or R¹—X

wherein: R¹ or R² is hydrogen, alkyl, alkenyl, alkynl, aromatic, cyclicor combinations thereof, X is any group containing carbon, sulfur,nitrogen, halogens, phosphorus, oxygen and combinations thereof.

While not limiting the invention in any way it is hypothesized that, asthe aqueous feed component evaporates during spray drying it leaves athin crust at the surface of the particle. The resulting particle wallor crust formed during the initial moments of spray drying appears totrap any high boiling blowing agents as hundreds of emulsion droplets(ca. 200-300 nm). As the drying process continues, the pressure insidethe particulate increases thereby vaporizing at least part of theincorporated blowing agent and forcing it through the relatively thincrust. This venting or outgassing apparently leads to the formation ofpores or other defects in the microstructure. At the same time remainingparticulate components (possibly including some blowing agent) migratefrom the interior to the surface as the particle solidifies. Thismigration apparently slows during the drying process as a result ofincreased resistance to mass transfer caused by an increased internalviscosity. Once the migration ceases the particle solidifies, leavingvoids, pores, defects, hollows, spaces, interstitial spaces, apertures,perforations or holes. The number of pores or defects, their size, andthe resulting wall thickness is largely dependent on the formulationand/or the nature of the selected blowing agent (e.g. boiling point),its concentration in the emulsion, total solids concentration, and thespray-drying conditions. As alluded to throughout the specification,this preferred particle morphology appears to contribute, at least inpart, to the improved powder dispersibility, suspension stability andaerodynamics.

It has been surprisingly found that substantial amounts of theserelatively high boiling blowing agents may be retained in the resultingspray dried product. That is, spray dried particulates as describedherein may comprise as much as 1%, 3%, 5%, 10%, 20%, 30% or even 40% w/wof residual blowing agent. In such cases, higher production yields wereobtained as a result an increased particle density caused by thisretained blowing agent. It will be appreciated by those skilled in theart that retained fluorinated blowing agent may alter the surfacecharacteristics of the particulates, thereby minimizing particleaggregation during processing and further increasing dispersionstability. Residual fluorinated blowing agent in the powders may alsoreduce the cohesive forces between particles by providing a barrier orby attenuating the attractive forces produced during manufacturing(e.g., electrostatics). This reduction in cohesive forces may beparticularly advantageous when using the disclosed microstructures inconjunction with dry powder inhalers.

Furthermore, the amount of residual blowing agent can be controlledthrough the process conditions (such as outlet temperature), blowingagent concentration, or boiling point. If the outlet temperature is ator above the boiling point, the blowing agent escapes the particle andthe production yield decreases. Preferred outlet temperature willgenerally be operated at 20, 30, 40, 50, 60, 70, 80, 90 or even 100° C.less than the blowing agent boiling point. More preferably thetemperature differential between the outlet temperature and the boilingpoint will range from 50 to 150° C. It will be appreciated by thoseskilled in the art that particle porosity, production yield,electrostatics and dispersibility can be optimized by first identifyingthe range of process conditions (e.g., outlet temperature) that aresuitable for the selected active agents and/or excipients. The preferredblowing agent can be then chosen using the maximum outlet temperaturesuch that the temperature differential with be at least 20 and up to150° C. In some cases, the temperature differential can be outside thisrange such as, for example, when producing the particulates undersupercritical conditions or using lyophilization techniques. Thoseskilled in the art will further appreciate that the preferredconcentration of blowing agent can be determined without undueexperimentation using techniques similar to those described in theExamples herein.

While residual blowing agent may be advantageous in selected embodimentsit may be desirable to substantially remove any blowing agent from thespray dried product. In this respect, the residual blowing agent caneasily be removed with a post-production evaporation step in a vacuumoven. Moreover, such post production techniques may be used to provideperforations in the particulates. For example, pores may be formed byspray drying a bioactive agent and an excipient that can be removed fromthe formed particulates under a vacuum.

In any event, typical concentrations of blowing agent in the feed stockare between 2% and 50% v/v, and more preferably between about 10% to 45%v/v. In other embodiments blowing agent concentrations will preferablybe greater than about 5%, 10%, 15%, 20%, 25% or even 30% v/v. Yet otherfeed stock emulsions may comprise 35%, 40%, 45% or even 50% v/v of theselected compound.

In preferred embodiments, another method of identifying theconcentration of blowing agent used in the feed is to provide it as aratio of the concentration of the blowing agent to that of thestabilizing surfactant (e.g. phosphatidylcholine or PC) in the precursoror feed emulsion. For fluorocarbon blowing agents (e.g. perfluorooctylbromide), and for the purposes of explanation, this ratio has beentermed the PFC/PC ratio. More generally, it will be appreciated thatcompatible blowing agents and/or surfactants may be substituted for theexemplary compounds without falling outside of the scope of the presentinvention. In any event, the typical PFC/PC ratio will range from about1 to about 60 and, more preferably, from about 10 to about 50. Forpreferred embodiments the ratio will generally be greater than about 5,10, 20, 25, 30, 40 or even 50. It should be appreciated that the use ofhigher PFC/PC ratios generally provides structures of a more hollow andporous nature. More particularly, those methods employing a PFC/PC ratioof greater than about 4.8 tended to provide structures that areparticularly compatible with the dry power formulations and dispersionsdisclosed herein.

While relatively high boiling point blowing agents comprise onepreferred aspect of the instant invention, it will be appreciated otherblowing or inflating agents may also be used to provide compatiblemicrostructures. As such, the blowing agent may comprise any volatilesubstance which can be incorporated into the feed solution for thepurpose of producing the desired microstructures. The blowing agent maybe removed during the initial drying process or during a post-productionstep such as vacuum drying or solvent extraction. Suitable agentsinclude:

1. Dissolved low-boiling (below 100° C.) agents miscible with aqueoussolutions, such as methylene chloride, acetone, ethyl acetate, andalcohols used to saturate the solution.

2. A gas, such as CO₂ or N₂, or liquid such as Freons, CFCs, HFAs, PFCs,HFCs, HFBs, fluoroalkanes and hydrocarbons, used at elevated pressure.

3. Emulsions of immiscible low-boiling (below 100° C.) liquids suitablefor use with the present invention are generally of the formula:

R¹—X—R² or R¹—X

 wherein: R¹ or R² is hydrogen, alkyl, alkenyl, alkynl, aromatic, cyclicor combinations thereof, X is any groups containing carbon, sulfur,nitrogen, halogens, phosphorus, oxygen and combinations thereof.

4. Dissolved or dispersed salts or organic substances which can beremoved under reduced pressure by sublimation in a post-production step,such as ammonium salts, camphor, etc.

5. Dispersed solids which can be extracted after the initial particlegeneration using a post-production solvent extraction step.

With respect to lower boiling point inflating agents, they are typicallyadded to the feed stock in quantities of about 1% to 40% v/v of thesurfactant solution. Approximately 15% v/v inflating agent has beenfound to produce a spray dried powder that may be used with the methodsof the present invention.

Regardless of which blowing agent is ultimately selected, it has beenfound that compatible particulates may be produced using commerciallyavailable equipment such as a Büchi mini spray drier (model B-191,Switzerland). As will be appreciated by those skilled in the art, theinlet temperature and the outlet temperature of the spray drier may beadjusted to provide the desired particle size and to maintain theactivity of the incorporated bioactive agent. In this regard, the inletand outlet temperatures are adjusted depending on the meltingcharacteristics of the formulation components and the composition of thefeed stock. The inlet temperature may thus be between 60° C. and 170°C., with the outlet temperatures of about 40° C. to 120° C. depending onthe composition of the feed and the desired particulate characteristics.Preferably these temperatures will be from 90° C. to 120° C. for theinlet and from 60° C. to 90° C. for the outlet. The flow rate which isused in the spray drying equipment will generally be about 3 ml perminute to about 15 ml per minute. The atomizer air flow rate will varybetween values of 25 liters per minute to about 50 liters per minute.Commercially available spray dryers are well known to those in the art,and suitable settings for any particular dispersion can be readilydetermined through standard empirical testing, with due reference to theexamples that follow.

Although the microparticulates are preferably formed using fluorinatedblowing agents in the form of an emulsion, it will be appreciated thatnonfluorinated oils may be used to increase the loading capacity of thebioactive agents without compromising the microstructure. In this case,selection of the nonfluorinated oil is based upon the solubility of theactive or bioactive agent, water solubility, boiling point, and flashpoint. The bioactive agent will be dissolved in the oil and subsequentlyemulsified in the feed solution. Preferably the oil will havesubstantial solubilization capacity with respect to the selected agent,low water solubility (<10³¹M), boiling point greater than water and aflash point greater than the drying outlet temperature. The addition ofsurfactants, and co-solvents to the nonfluorinated oil to increase thesolubilization capacity is also within the scope of the presentinvention.

In particularly preferred embodiments nonfluorinated oils may be used tosolubilize bioactive agents that have limited solubility in aqueouscompositions. The use of nonfluorinated oils is of particular use forincreasing the loading capacity of hydrophobic peptides and proteins.Preferably the oil or oil mixture for solubilizing these compounds willhave a refractive index between 1.36 and 1.41 (e.g. ethyl butyrate,butyl carbonate, dibutyl ether). In addition, process conditions, suchas temperature and pressure, may be adjusted in order to boostsolubility of the selected agent. It will be appreciated that selectionof an appropriate oil or oil mixtures and processing conditions tomaximize the loading capacity of an agent are well within the purview ofa skilled artisan in view of the teachings herein and may beaccomplished without undue experimentation.

Particularly preferred embodiments of the present invention comprisespray drying preparations comprising a surfactant such as a phospholipidand at least one bioactive agent. Other embodiments include spray dryingpreparations that may further include an excipient comprising ahydrophilic moiety such as, for example, a carbohydrate (i.e. glucose,lactose, or starch) in addition to any selected surfactant. In thisregard, various starches and derivatized starches are particularlysuitable for use in the present invention. Other optional components mayinclude conventional viscosity modifiers, buffers such as phosphatebuffers or other conventional biocompatible buffers or pH adjustingagents such as acids or bases, and osmotic agents (to provideisotonicity, hyperosmolarity, or hyposmolarity). Examples of suitablesalts include sodium phosphate (both monobasic and dibasic), sodiumchloride, calcium phosphate, calcium chloride and other physiologicallyacceptable salts.

Whatever components are selected, the first step in particulateproduction typically comprises feed stock preparation. Preferably theselected drug is dissolved in water to produce a concentrated solution.The drug may also be dispersed directly in the emulsion, particularly inthe case of water insoluble agents. Alternatively, the drug may beincorporated in the form of a solid particulate dispersion. Theconcentration of the active or bioactive agent used is dependent on theamount of agent required in the final powder and the performance of thedelivery device employed (e.g., the fine particle dose for a MDI orDPI). As needed, cosurfactants such as poloxamer 188 or span 80 may bedispersed into this annex solution. Additionally, excipients such assugars and starches can also be added.

In selected embodiments an oil-in-water emulsion is then formed in aseparate vessel. The oil employed is preferably a fluorocarbon (e.g.,perfluorooctyl bromide, perfluorodecalin) which is emulsified using asurfactant such as a long chain saturated phospholipid. For example, onegram of phospholipid may be homogenized in 150 g hot distilled water(e.g., 60° C.) using a suitable high shear mechanical mixer (e.g.,Ultra-Turrax model T-25 mixer) at 8000 rpm for 2 to 5 minutes. Typically5 to 25 g of fluorocarbon is added dropwise to the dispersed surfactantsolution while mixing. The resulting perfluorocarbon in water emulsionis then processed using a high pressure homogenizer to reduce theparticle size. For example, the emulsion may be processed at 12,000 to18,000 psi, 5 discrete passes and kept at 50 to 80° C.

The bioactive agent solution and perfluorocarbon emulsion may then becombined and fed into the spray dryer. Typically the two preparationswill be miscible as the emulsion will preferably comprise an aqueouscontinuous phase. While the bioactive agent is solubilized separatelyfor the purposes of the instant discussion it will be appreciated that,in other embodiments, the active or bioactive agent may be solubilized(or dispersed) directly in the emulsion. In such cases, the active orbioactive emulsion is simply spray dried without combining a separatedrug preparation.

In any event, operating conditions such as inlet and outlet temperature,feed rate, atomization pressure, flow rate of the drying air, and nozzleconfiguration can be adjusted in accordance with the manufacturer'sguidelines in order to produce the required particle size, andproduction yield of the resulting dry microstructures. Exemplarysettings are as follows: an air inlet temperature between 60° C. and170° C.; an air outlet between 40° C. to 120° C.; a feed rate between 3ml to about 15 ml per minute; and an aspiration air flow of 300 L/min.and an atomization air flow rate between 25 to 50 L/min. The selectionof appropriate apparatus and processing conditions are well within thepurview of a skilled artisan in view of the teachings herein and may beaccomplished without undue experimentation. In any event, the use ofthese and substantially equivalent methods provide for the formation ofhollow porous aerodynamically light microspheres with particle diametersappropriate for aerosol deposition into the lung, microstructures thatare both hollow and porous, almost honeycombed or foam-like inappearance. In especially preferred embodiments the perforatedmicrostructures comprise hollow, porous spray dried microspheres.

Along with spray drying, perforated microstructures useful in thepresent invention may be formed by lyophilization. Those skilled in theart will appreciate that lyophilization is a freeze-drying process inwhich water is sublimed from the composition after it is frozen. Theparticular advantage associated with the lyophilization process is thatbiologics and other pharmaceuticals that are relatively unstable in anaqueous solution can be dried without elevated temperatures (therebyeliminating the adverse thermal effects), and then stored in a dry statewhere there are few stability problems. With respect to the instantinvention such techniques are particularly compatible with theincorporation of peptides, proteins, genetic material and other naturaland synthetic macromolecules in particulates or perforatedmicrostructures without compromising physiological activity. Methods forproviding lyophilized particulates are known to those of skill in theart and it would clearly not require undue experimentation to providecompatible microstructures in accordance with the teachings herein. Thelyophilized cake containing a fine foam-like structure can be micronizedusing techniques known in the art to provide particles having meandiameters under 5 μm or 10 μm. Accordingly, to the extent thatlyophilization processes may be used to provide microstructures havingthe desired characteristics they are expressly contemplated as beingwithin the scope of the instant invention.

Besides the aforementioned techniques, the particulates and perforatedmicrostructures of the present invention may also be formed using amethod where a feed solution (either emulsion or aqueous) containingwall forming agents is rapidly added to a reservoir of heated oil (e.g.perflubron or other high boiling FCs) under reduced pressure. The waterand volatile solvents of the feed solution rapidly boils and areevaporated. This process may be used to provide a perforated structurefrom wall forming agents similar to puffed rice or popcorn. Preferablythe wall forming agents are insoluble in the heated oil. The resultingparticles can then separated from the heated oil using a filteringtechnique and subsequently dried under vacuum.

Additionally, the particles or perforated microstructures of the presentinvention may also be formed using a double emulsion method. In thedouble emulsion method the medicament is first dispersed in a polymerdissolved in an organic solvent (e.g. methylene chloride) by sonicationor homogenization. This primary emulsion is then stabilized by forming amultiple emulsion in a continuous aqueous phase containing an emulsifiersuch as polyvinylalcohol. Evaporation or extraction using conventionaltechniques and apparatus then removes the organic solvent. The resultingmicroparticles are then washed, filtered and dried prior to use orcombining them w ith an appropriate suspension medium in accordance withthe present invention.

F. Administration

Whatever method is ultimately selected for production of themicroparticulates, the resulting powders have a number of advantageousproperties that allow them to be effectively used in either a powderedform or as a dispersion comprising a nonaqueous suspension medium. Inparticularly preferred embodiments the bioactive compositions, whetherin the form of a dry powder or dispersion, will be administered to themucosal surface of the respiratory tract (i.e., the pulmonary and/or thenasal tract) via inhalation therapy. Such administration may be effectedusing MDIs, DPIs, nebulizers, nasal pumps, atomizers, spray bottles orby direct instillation in the form of drops. However, while inhalationtherapies are extremely compatible with the present invention, it willbe appreciated that other forms and/or routes of administration are alsouseful.

In this regard, the powders and stabilized dispersions of the presentinvention may also be used for the localized or systemic administrationof compounds to any location of the body. Accordingly, it should beemphasized that, in preferred embodiments, the preparations may beadministered using a number of different routes including, but notlimited to, topical, intramuscular, transdermal, intradermal,intraperitoneal, nasal, pulmonary, buccal, vaginal, rectal, aural, oralor ocular administration. Preferred target sites may be found in, forexample, the gastrointestinal tract, urogenital tract or respiratorytract. More generally, the stabilized dispersions of the presentinvention may be used to deliver agents topically or by administrationto any body cavity. In preferred embodiments the body cavity is selectedfrom the group consisting of the peritoneum, sinus cavity, rectum,urethra, stomach, nasal cavity, vagina, auditory meatus, oral cavity,buccal pouch and pleura. Those skilled in the art will appreciate thatthe selected route of administration will largely be determined by thechoice of bioactive agent and the desired response of the subject.

With regard to the delivery of the disclosed powders or stabilizeddispersions, another aspect of the present invention is directed tosystems for the administration of one or more bioactive agents orbiologics to a patient. As alluded to above, exemplary inhalationdevices compatible with the present invention may comprise an atomizer,nasal pump, a sprayer or spray bottle, a dry powder inhaler, a metereddose inhaler or a nebulizer. In preferred embodiments, these inhalationsystems will deliver the bioactive agent to the desired physiologicalsite (e.g. a mucosal surface) as an aerosol. For the purposes of theinstant application the term “aerosolized” shall be held to mean agaseous suspension of fine solid or liquid particles unless otherwisedictated by contextual restraints. That is, an aerosol or aerosolizedmedicament may be generated, for example, by a dry powder inhaler, ametered dose inhaler, an atomizer, a spray bottle or a nebulizer. Ofcourse, as explained in more detail below, the compositions of thepresent invention may also be delivered directly (e.g. by conventionalinjection or needleless injection) or using such techniques as liquiddose instillation. As such, a further aspect of the present invention isdirected to needleless injectors (e.g. pressurized gas guns) comprisingthe disclosed powders or dispersions.

F(i). Dry Powder Inhalers

With respect to inhalation therapies, those skilled in the art willappreciate that the powders of the present invention, particularly thosecomprising perforated microstructures, are particularly useful in DPIs.Conventional DPIs, or dry powder inhalers, comprise powderedformulations and devices where a predetermined dose of medicament,either alone or in a blend with lactose carrier particles, is deliveredas a fine mist or aerosol of dry powder for inhalation. Useful DPImedicaments are typically formulated so that they readily disperse intodiscrete particles with a size rage between 0.5 to 20 μm. The powder isactuated either by inspiration or by some external delivery force, suchas pressurized air. DPI formulations are typically packaged in singledose units or they employ reservoir systems capable of metering multipledoses with manual transfer of the dose to the device.

DPIs are generally classified based on the dose delivery systememployed. In this respect, the two major types of DPIs comprise unitdose delivery devices and bulk reservoir delivery systems. As usedherein, the term “reservoir” shall be used in a general sense and heldto encompass both configurations unless otherwise dictated by contextualrestraints. In any event, unit dose delivery systems require the dose ofpowder formulation presented to the device as a single unit. With thissystem, the formulation is prefilled into dosing wells which may befoil-packaged or presented in blister strips to prevent moistureingress. Other unit dose packages include hard gelatin capsules. Mostunit dose containers designed for DPIs are filled using a fixed volumetechnique. As a result, there are physical limitations (here density) tothe minimal dose that can be metered into a unit package, which isdictated by the powder flowability and bulk density.

As previously alluded to, the powders of the present invention obviatemany of the difficulties associated with prior art carrier preparations.That is, an improvement in DPI performance may be provided by adjustingthe particle size, aerodynamics, morphology and density, humidity andcharge as disclosed herein. In this respect the present inventionprovides for formulations wherein the medicament and the incipients orbulking agents are preferably associated with or comprise perforatedmicrostructures. As set forth above, preferred compositions according tothe present invention typically yield powders with bulk densities lessthan 0.1 g/cm³ and often less than 0.05 g/cm³. It will be appreciatedthat providing powders having bulk densities an order of a magnitudeless than conventional DPI formulations allows for much lower doses ofthe selected bioactive agent to be filled into a unit dose container ormetered via reservoir-based DPIs. The ability to effectively meter smallquantities is significant for relatively potent bioactive agents such ashormones. Moreover, the ability to effectively deliver particulateswithout associated carrier particles simplifies product formulation,filling and reduces undesirable side effects.

It will be appreciated that the powders of the present invention areparticularly effective at delivering relatively high doses of bioactiveagent in a single actuation. Unlike prior art formulations, the powderedformulations do not require the use of bulking agents for effectivefilling and delivery and may therefore comprise higher levels ofbioactive agent on a weight by weight basis. Significantly, thedisclosed compositions may be used to deliver as much as approximately10 mg of bioactive agent in a single actuation. Such advantages may beparticularly important when delivering, for example, immunomodulators orantibodies for passive immunization, that may not be as potent as othercompatible agents. Of course, while the instant discussion isspecifically directed to the use of DPIs, this same advantage is equallyapplicable to dispersion formulations and other forms of administrationsuch as MDIs, nasal pumps and needleless injectors.

In addition to the aforementioned advantages, preferred embodiments ofthe present invention exhibit favorable aerodynamic properties that makethem particularly effective for use in DPIs. More specifically, theperforated structure and relatively high surface area of themicroparticles enables them to be carried along in the flow of gasesduring inhalation with greater ease and for longer distances thanrelatively non-perforated particles of comparable size. Because of theirhigh porosity and low density, administration of perforatedmicrostructures with a DPI provides for increased particle deposition attarget sites such as mucosal surfaces in the nasal passages andperipheral regions of the lung with correspondingly less deposition inthe throat. Such particle distribution may be employed to increase thedeep lung deposition of the administered agent that is preferable forsystemic administration. Moreover, in a substantial improvement overprior art DPI preparations the low-density, highly porous powders of thepresent invention preferably eliminate the need for carrier particles clF(ii). Stabilized Dispersions

Along with their use in a dry powder configuration, it will beappreciated that the powders of the present invention may beincorporated in a suspension medium to provide stabilized dispersions.Preferably, the stabilized dispersions will comprise a nonaqueoussuspension medium. Among other uses, the stabilized dispersions providefor the effective delivery of bioactive agents to the pulmonary airpassages of a patient using MDIs, atomizers or spray bottles, nasalpumps, needleless injectors, nebulizers or liquid dose instillation (LDItechniques).

Those skilled in the art will appreciate the enhanced stability of thedisclosed dispersions or suspensions is largely achieved by lowering thevan der Waals attractive forces between the suspended particles, and byreducing the differences in density between the suspension medium andthe particles. In accordance with the teachings herein, the increases insuspension stability may be imparted by engineering perforatedmicrostructures which are then dispersed in a compatible suspensionmedium. As discussed above, the perforated microstructures comprisepores, voids, hollows, defects or other interstitial spaces that allowthe fluid suspension medium to freely permeate or perfuse theparticulate boundary. Particularly preferred embodiments compriseperforated microstructures that are both hollow and porous, almosthoneycombed or foam-like in appearance. In especially preferredembodiments the perforated microstructures comprise hollow, porous spraydried microspheres. Of course, in other embodiments, including thosecomprising relatively nonporous, solid particulates, enhanced stabilitymay be imparted through the selection of particulate components (e.g.surfactants).

When perforated microstructures are placed in the suspension medium(i.e. a hydrofluoroalkane propellant or liquid fluorocarbon), thesuspension medium is able to permeate the particles, thereby creating a“homodispersion”, wherein both the continuous and dispersed phases areindistinguishable. Since the defined or “virtual” particles (i.e.comprising the volume circumscribed by the microparticulate matrix) aremade up almost entirely of the medium in which they are suspended, theforces driving particle aggregation (flocculation) are minimized.Additionally, the differences in density between the defined particlesand the continuous phase are minimized by having the microstructuresfilled with the medium, thereby effectively slowing particle creaming orsedimentation. As such, the particulates and stabilized suspensions ofthe present invention are particularly compatible with manyaerosolization techniques, such as MDI, atomization via a spray bottle,nasal pumps, nebulization and the like. Moreover, the stabilizeddispersions are compatible with other routes of administrationincluding, but not limited to, liquid dose instillation, needlelessinjection, conventional injection and topical applications.

Unlike prior art compositions, preferred suspensions of the instantinvention are designed not to increase repulsion between particles, butrather to decrease the attractive forces between particles. In thisrespect it should be appreciated that the principal forces drivingflocculation in nonaqueous media are van der Waals attractive forces. Asdiscussed above, VDW forces are quantum mechanical in origin, and can bevisualized as attractions between fluctuating dipoles (i.e. induceddipole-induced dipole interactions). Dispersion forces are extremelyshort-range and scale as the sixth power of the distance between atoms.When two macroscopic bodies approach one another the dispersionattractions between the atoms sums up. The resulting force is ofconsiderably longer range, and depends on the geometry of theinteracting bodies.

More specifically, for two spherical particles, the magnitude of the VDWpotential, V_(A), can be approximated by${V_{A} = {\frac{- A_{eff}}{6H_{0}}\quad \frac{R_{1}\quad R_{2}}{( {R_{1} + R_{2}} )}}},$

where A_(eff) is the effective Hamaker constant which accounts for thenature of the particles and the medium, H₀ is the distance betweenparticles, and R₁ and R₂ are the radii of spherical particles 1 and 2.The effective Hamaker constant is proportional to the difference in thepolarizabilities of the dispersed particles and the suspension medium:A_(eff)=({square root over (A_(SM))}−{square root over (A_(PART))})²,where A_(SM) and A_(PART) are the Hamaker constants for the suspensionmedium and the particles, respectively. As the suspended particles andthe dispersion medium become similar in nature, A_(SM) and A_(PART)become closer in magnitude, and A_(eff) and V_(A) become smaller. Thatis, by reducing the differences between the Hamaker constant associatedwith suspension medium and the Hamaker constant associated with thedispersed particles, the effective Hamaker constant (and correspondingvan der Waals attractive forces) may be reduced.

One way to minimize the differences in the Hamaker constants is tocreate a “homodispersion”, that is make both the continuous anddispersed phases essentially indistinguishable as discussed above.Besides exploiting the morphology of the particles to reduce theeffective Hamaker constant, the components of the structural matrix(defining the perforated microstructures) will preferably be chosen soas to exhibit a Hamaker constant relatively close to that of theselected suspension medium. In this respect, one may use the actualvalues of the Hamaker constants of the suspension medium and theparticulate components to determine the compatibility of the dispersioningredients and provide a good indication as to the stability of thepreparation. Alternatively, one could select relatively compatibleparticulate or perforated microstructure components and suspensionmediums using characteristic physical values that coincide withmeasurable Hamaker constants but are more readily discernible.

In this respect, it has been found that the refractive index values ofmany compounds tend to scale with the corresponding Hamaker constant.Accordingly, easily measurable refractive index values may be used toprovide a fairly good indication as to which combination of suspensionmedium and particle excipients will provide a dispersion having arelatively low effective Hamaker constant and associated stability. Itwill be appreciated that, since refractive indices of compounds arewidely available or easily derived, the use of such values allows forthe formation of stabilized dispersions in accordance with the presentinvention without undue experimentation. For the purpose of illustrationonly, the refractive indices of several compounds compatible with thedisclosed dispersions are provided in Table I immediately below:

TABLE I Compound Refractive Index HFA-134a 1.172 HFA-227 1.223 CFC-121.287 CFC-114 1.288 PFOB 1.305 Mannitol 1.333 Ethanol 1.361 n-octane1.397 DMPC 1.43 Pluronic F-68 1.43 Sucrose 1.538 Hydroxyethylstarch 1.54Sodium chloride 1.544

Consistent with the compatible dispersion components set forth above,those skilled in the art will appreciate that, the formation ofdispersions wherein the components have a refractive index differentialof less than about 0.5 is preferred. That is, the refractive index ofthe suspension medium will preferably be within about 0.5 of therefractive index associated with the particles or perforatedmicrostructures. It will further be appreciated that, the refractiveindex of the suspension medium and the particles may be measureddirectly or approximated using the refractive indices of the majorcomponent in each respective phase. For the particulates or perforatedmicrostructures, the major component may be determined on a weightpercent basis. For the suspension medium, the major component willtypically be derived on a volume percentage basis. In selectedembodiments of the present invention the refractive index differentialvalue will preferably be less than about 0.45, about 0.4, about 0.35 oreven less than about 0.3. Given that lower refractive indexdifferentials imply greater dispersion stability, particularly preferredembodiments comprise index differentials of less than about 0.28, about0.25, about 0.2, about 0.15 or even less than about 0.1. It is submittedthat a skilled artisan will be able to determine which excipients areparticularly compatible without undue experimentation given the instantdisclosure. The ultimate choice of preferred excipients will also beinfluenced by other factors, including biocompatibility, regulatorystatus, ease of manufacture, cost.

As discussed above, minimization of density differences between theparticles and the continuous phase may be achieved by using perforatedand/or hollow microstructures, such that the suspension mediumconstitutes most of the particle volume. As used herein, the term“particle volume” corresponds to the volume of suspension medium thatwould be displaced by incorporated hollow/porous particles if they weresolid, i.e. the volume defined by the particle boundary. For thepurposes of explanation, and as discussed above, these fluid filledparticulate volumes may be referred to as “virtual particles.”Preferably, the average volume of the bioactive agent/excipient shell ormatrix (i.e. the volume of medium actually displaced by the perforatedmicrostructure) comprises less than 80% of the average particle volume(or less than 80% of the virtual particle). More preferably, the volumeof the microparticulate matrix comprises less than about 50%, 40%, 30%or even 20% of the average particle volume. Even more preferably, theaverage volume of the shell/matrix comprises less than about 10%, 5%, 3%or 1% of the average particle volume. Those skilled in the art willappreciate that such matrix or shell volumes typically contribute littleto the virtual particle density which is overwhelmingly dictated by thesuspension medium found therein.

It will further be appreciated that, the use of such microstructureswill allow the apparent density of the virtual particles to approachthat of the suspension medium substantially eliminating the attractivevan der Waals forces. Moreover, as previously discussed, the componentsof the microparticulate matrix are preferably selected, as much aspossible given other considerations, to approximate the density ofsuspension medium. Accordingly, in preferred embodiments of the presentinvention, the virtual particles and the suspension medium will have adensity differential of less than about 0.6 g/cm³. That is, the meandensity of the virtual particles (as defined by the matrix boundary)will be within approximately 0.6 g/cm³ of the suspension medium. Morepreferably, the mean density of the virtual particles will be within0.5, 0.4, 0.3 or 0.2 g/cm³ of the selected suspension medium. In evenmore preferable embodiments the density differential will be less thanabout 0.1, 0.05, 0.01, or even less than 0.005 g/cm³.

In addition to the aforementioned advantages, the use of the disclosedparticulates allows for the formation of dispersions comprising muchhigher volume fractions of particles in suspension. It should beappreciated that, the formulation of prior art dispersions at volumefractions approaching close-packing generally results in dramaticincreases in dispersion viscoelastic behavior. Rheological behavior ofthis type is not appropriate for MDI or nebulizer applications. Thoseskilled in the art will appreciate that, the volume fraction of theparticles may be defined as the ratio of the apparent volume of theparticles (i.e. the particle volume) to the total volume of the system.Each system has a maximum volume fraction or packing fraction. Forexample, particles in a simple cubic arrangement reach a maximum packingfraction of 0.52 while those in a face centered cubic/hexagonal closepacked configuration reach a maximum packing fraction of approximately0.74. For non-spherical particles or polydisperse systems, the derivedvalues are different. Accordingly, the maximum packing fraction is oftenconsidered to be an empirical parameter for a given system.

Here, it was surprisingly found that the preferred particulates of thepresent invention do not exhibit undesirable viscoelastic behavior evenat high volume fractions, approaching close packing. To the contrary,they remain as free flowing, low viscosity suspensions having little orno yield stress when compared with analogous suspensions comprisingsolid particulates. The low viscosity of the disclosed suspensions isthought to be due, at least in large part, to the relatively low van derWaals attraction between the fluid-filled hollow, porous particles. Assuch, in selected embodiments the volume fraction of the discloseddispersions is greater than approximately 0.3. Other embodiments mayhave packing values on the order of 0.3 to about 0.5 or on the order of0.5 to about 0.8, with the higher values approaching a close packingcondition. Moreover, as particle sedimentation tends to naturallydecrease when the volume fraction approaches close packing, theformation of relatively concentrated dispersions may further increaseformulation stability.

Although the methods and compositions of the present invention may beused to form relatively concentrated suspensions, the stabilizingfactors work equally well at much lower packing volumes and suchdispersions are contemplated as being within the scope of the instantdisclosure. In this regard, it will be appreciated that, dispersionscomprising low volume fractions are extremely difficult to stabilizeusing prior art techniques. Conversely, dispersions incorporatingparticulates comprising a bioactive agent as described herein areparticularly stable even at low volume fractions. Accordingly, thepresent invention allows for stabilized dispersions, and particularlyrespiratory dispersions, to be formed and used at volume fractions lessthan 0.3. In some preferred embodiments, the volume fraction isapproximately 0.0001-0.3, more preferably 0.001-0.01. Yet otherpreferred embodiments comprise stabilized suspensions having volumefractions from approximately 0.01 to approximately 0.1.

The perforated microstructures of the present invention may also be usedto stabilize dilute suspensions of micronized bioactive agents. In suchembodiments the perforated microstructures may be added to increase thevolume fraction of particles in the suspension, thereby increasingsuspension stability to creaming or sedimentation. Further, in theseembodiments the incorporated microstructures may also act in preventingclose approach (aggregation) of the micronized drug particles. It shouldbe appreciated that, the perforated microstructures incorporated in suchembodiments do not necessarily comprise a bioactive agent. Rather, theymay be formed exclusively of various excipients, including surfactants.

Those skilled in the art will further appreciate that the stabilizedsuspensions or dispersions of the present invention may be prepared bydispersal of the microstructures in the selected suspension medium whichmay then be placed in a container or reservoir. In this regard, thestabilized preparations of the present invention can be made by simplycombining the components in sufficient quantity to produce the finaldesired dispersion concentration. Although the microstructures readilydisperse without mechanical energy, the application of mechanical energyto aid in dispersion (e.g. with the aid of sonication) is contemplated.Alternatively, the components may be mixed by simple shaking or othertype of agitation. The process is preferably carried out under anhydrousconditions to obviate any adverse effects of moisture on suspensionstability. Once formed, the dispersion has a reduced susceptibility toflocculation and sedimentation.

As indicated throughout the instant specification, the dispersions ofthe present invention are preferably stabilized. In a broad sense, theterm “stabilized dispersion” will be held to mean any dispersion thatresists aggregation, flocculation or creaming to the extent required toprovide for the effective delivery of a bioactive agent. Moreover, it isa significant advantage of the instant invention that the discloseddispersions and powders are stable at room temperature and do notrequire refrigeration or freezing to effectively maintain theiractivity. Besides prolonging shelf life, this remarkable temperaturestability greatly simplifies shipping and administration.

While those skilled in the art will appreciate that there are severalmethods that may be used to assess the stability of a given dispersion,a preferred method for the purposes of the present invention comprisesdetermination of creaming or sedimentation time using a dynamicphotosedimentation method. A preferred method comprises subjectingsuspended particles to a centrifugal force and measuring absorbance ofthe suspension as a function of time. A rapid decrease in the absorbanceidentifies a suspension with poor stability. It is submitted that thoseskilled in the art will be able to adapt the procedure to specificsuspensions without undue experimentation.

For the purposes of the present invention the creaming time shall bedefined as the time for the suspended drug particulates to cream to ½the volume of the suspension medium. Similarly, the sedimentation timemay be defined as the time it takes for the particulates to sediment in½ the volume of the liquid medium. Besides the photosedimentationtechnique described above, a relatively simple way to determine thecreaming time of a preparation is to provide the particulate suspensionin a sealed glass vial. The vials are agitated or shaken to providerelatively homogeneous dispersions which are then set aside and observedusing appropriate instrumentation or by visual inspection. The timenecessary for the suspended particulates to cream to ½ the volume of thesuspension medium (i.e., to rise to the top half of the suspensionmedium), or to sediment within ½ the volume (i.e., to settle in thebottom ½ of the medium), is then noted. Suspension formulations having acreaming time greater than 1 minute are preferred and indicate suitablestability. More preferably, the stabilized dispersions comprise creamingtimes of greater than 1, 2, 5, 10, 15, 20 or 30 minutes. In particularlypreferred embodiments, the stabilized dispersions exhibit creaming timesof greater than about 1, 1.5, 2, 2.5, or 3 hours. Substantiallyequivalent periods for sedimentation times are indicative of compatibledispersions.

It will also be understood that other components can be included in thestabilized dispersions of the present invention. For example, osmoticagents, stabilizers, chelators, buffers, viscosity modulators, salts,and sugars can be added to fine tune the stabilized dispersions formaximum life and ease of administration. Such components may be addeddirectly to the suspension medium or associated with, or incorporatedin, the perforated microstructures. Considerations such as sterility,isotonicity, and biocompatibility may govern the use of conventionaladditives to the disclosed compositions. The use of such agents will beunderstood to those of ordinary skill in the art and, the specificquantities, ratios, and types of agents can be determined empiricallywithout undue experimentation.

F(iii). Metered Dose Inhalers

As discussed, the stabilized dispersions may preferably be administeredto the nasal or pulmonary air passages of a patient via aerosolization,such as with a metered dose inhaler. The use of such stabilizedpreparations provides for superior dose reproducibility and improveddeposition at the target site as described above. MDIs are well known inthe art and could easily be employed for administration of the claimeddispersions without undue experimentation. Breath activated MDIs, aswell as those comprising other types of improvements which have been, orwill be, developed are also compatible with the stabilized dispersionsand present invention and, as such, are contemplated as being with inthe scope thereof.

MDI canisters generally comprise a container or reservoir capable ofwithstanding the vapor pressure of the propellant used such as, aplastic or plastic-coated glass bottle, or preferably, a metal can or,for example, an aluminum can which may optionally be anodized,lacquer-coated and/or plastic-coated, wherein the container is closedwith a metering valve. The metering valves are designed to deliver ametered amount of the formulation per actuation. The valves incorporatea gasket to prevent leakage of propellant through the valve. The gasketmay comprise any suitable elastomeric material such as, for example, lowdensity polyethylene, chlorobutyl, black and whitebutadiene-acrylonitrile rubbers, butyl rubber and neoprene. Suitablevalves are commercially available from manufacturers well known in theaerosol industry, for example, from Valois, France (e.g. DFIO, DF30, OF31/50 ACT, DF60), Bespak plc, LTK (e.g. BK300, BK356) and 3M-NeotechnicLtd., LIK (e.g. Spraymiser).

Each filled canister is conveniently fitted into a suitable channelingdevice or actuator prior to use to form a metered dose inhaler foradministration of the medicament into the lungs or oral or nasal cavityof a patient. Suitable channeling devices comprise for example a valveactuator and a cylindrical or cone-like passage through which medicamentmay be delivered from the filled canister via the metering valve, to thenose or mouth of a patient e.g., a mouthpiece actuator. Metered doseinhalers are designed to deliver a fixed unit dosage of medicament peractuation such as, for example, in the range of 10 to 5000 micrograms ofbioactive agent per actuation. Typically, a single charged canister willprovide for tens or even hundreds of shots or doses.

With respect to MDIs, it is an advantage of the present invention thatany biocompatible suspension medium having adequate vapor pressure toact as a propellant may be used. Particularly preferred suspension mediaare compatible with use in a metered dose inhaler. That is, they will beable to form aerosols upon the activation of the metering valve andassociated release of pressure. In general, the selected suspensionmedium should be biocompatible (i.e. relatively non-toxic) andnon-reactive with respect to the suspended perforated microstructurescomprising the bioactive agent. Preferably, the suspension medium willnot act as a substantial solvent for any components incorporated in theperforated microspheres. Selected embodiments of the invention comprisesuspension media selected from the group consisting of fluorocarbons(including those substituted with other halogens), hydrofluoroalkanes,perfluorocarbons, hydrocarbons, alcohols, ethers or combinationsthereof. It will be appreciated that, the suspension medium may comprisea mixture of various compounds selected to impart specificcharacteristics.

Particularly suitable propellants for use in the MDI suspension mediumsof the present invention are those propellant gases that can beliquefied under pressure at room temperature and, upon inhalation ortopical use, are safe, toxicologically innocuous and free of sideeffects. In this regard, compatible propellants may comprise anyhydrocarbon, fluorocarbon, hydrogen-containing fluorocarbon or mixturesthereof having a sufficient vapor pressure to efficiently form aerosolsupon activation of a metered dose inhaler. Those propellants, typicallytermed hydrofluoroalkanes or HFAs, are especially compatible. Suitablepropellants include, for example, short chain hydrocarbons, C₁₋₄hydrogen-containing chlorofluorocarbons such as CH₂ClF, CCl₂F₂CHClF,CF₃CHClF, CHF₂CClF₂, CHClFCHF₂, CF₃CH₂Cl, and CClF₂CH₃; C₁₋₄hydrogen-containing fluorocarbons (e.g. HFAs) such as CHF₂CHF₂, CF₃CH₂F,CHF₂CH₃, and CF₃CHFCF₃; and perfluorocarbons such as CF₃CF₃ andCF₃CF₂CF₃. Preferably, a single perfluorocarbon or hydrogen-containingfluorocarbon is employed as the propellant. Particularly preferred aspropellants are 1,1,1,2-tetrafluoroethane (CF₃CH₂F) (HFA-134a) and1,1,1,2,3,3,3-heptafluoro-n-propane (CF₃CHFCF₃) (HFA-227),perfluoroethane, monochlorodifluoromethane, 1,1-difluoroethane, andcombinations thereof. It is desirable that the formulations contain nocomponents that deplete stratospheric ozone. In particular it isdesirable that the formulations are substantially free ofchlorofluorocarbons such as CCl₃F, CCl₂F₂, and CF₃CCl₃.

While preferred embodiments of the invention comprise ecologicallybenign suspension media, traditional chlorofluorocarbons and substitutedfluorinated compounds may also be used as suspension mediums inaccordance with the teachings herein. In this respect, FC-11 (CCL3F),FC-11B1 (CBrCl2F), FC-11B2 (CBr2ClF), FC12B2 (CF2Br2), FC21 (CHCl2F),FC21B1 (CHBrClF), FC-21B2 (CHBr2F), FC-31B1 (CH2BrF), FC113A (CCl3CF3),FC-122 (CClF2CHCl2), FC-123 (CF3CHCl2), FC-132 (CHClFCHClF), FC-133(CHClFCHF2), FC-141 (CH2ClCHClF), FC-141B (CCl2FCH3), FC-142(CHF2CH2Cl), FC-151 (CH2FCH2Cl), FC-152 (CH2FCH2F), FC-1112 (CClF═CClF),FC-1121 (CHCl═CFCl) and FC-1131 (CHCl═CHF) are all compatible with theteachings herein despite possible attendant environmental concerns. Assuch, each of these compounds may be used, alone or in combination withother compounds (i.e. less volatile fluorocarbons) to form stabilizedrespiratory dispersions in accordance with the present invention.

F(iv). Nebulizers

Along with the aforementioned embodiments, the stabilized dispersions ofthe present invention may also be used in conjunction with nebulizers toprovide an aerosolized medicament that may be administered to thepulmonary air passages of a patient in need thereof. Nebulizers are wellknown in the art and could easily be employed for administration of theclaimed dispersions without undue experimentation. Breath activatednebulizers, as well as those comprising other types of improvementswhich have been, or will be, developed are also compatible with thestabilized dispersions and present invention and are contemplated asbeing with in the scope thereof.

Nebulizers work by forming aerosols, that is converting a bulk liquidinto small droplets suspended in a breathable gas. Here, the aerosolizedmedicament to be administered (preferably to the pulmonary air passages)will comprise small droplets of suspension medium associated withperforated microstructures comprising a bioactive agent. In suchembodiments, the stabilized dispersions of the present invention willtypically be placed in a fluid reservoir operably associated with anebulizer. The specific volumes of preparation provided, means offilling the reservoir, etc., will largely be dependent on the selectionof the individual nebulizer and is well within the purview of theskilled artisan. Of course, the present invention is entirely compatiblewith single-dose nebulizers and multiple dose nebulizers.

The present invention overcomes these and other difficulties byproviding stabilized dispersions with a suspension medium thatpreferably comprises a fluorinated compound (i.e. a fluorochemical,fluorocarbon or perfluorocarbon). Particularly preferred embodiments ofthe present invention comprise fluorochemicals that are liquid at roomtemperature. As indicated above, the use of such compounds, whether as acontinuous phase or, as a suspension medium, provides several advantagesover prior art liquid inhalation preparations. In this regard, it iswell established that many fluorochemicals have a proven history ofsafety and biocompatibility in the lung. Further, in contrast to aqueoussolutions, fluorochemicals do not negatively impact gas exchangefollowing pulmonary administration. To the contrary, they may actuallybe able to improve gas exchange and, due to their unique wettabilitycharacteristics, are able to carry an aerosolized stream of particlesdeeper into the lung, thereby improving systemic delivery of the desiredpharmaceutical compound. In addition, the relatively non-reactive natureof fluorochemicals acts to retard any degradation (by proteolysis orhydrolysis) of an incorporated bioactive agent.

In any event, nebulizer mediated aerosolization typically requires aninput of energy in order to produce the increased surface area of thedroplets and, in some cases, to provide transportation of the atomizedor aerosolized medicament. One common mode of aerosolization is forcinga stream of fluid to be ejected from a nozzle, whereby droplets areformed. With respect to nebulized administration, additional energy isusually imparted to provide droplets that will be sufficiently small tobe transported deep into the lungs. Thus, additional energy is needed,such as that provided by a high velocity gas stream or a piezoelectriccrystal. Two popular types of nebulizers, jet nebulizers and ultrasonicnebulizers, rely on the aforementioned methods of applying additionalenergy to the fluid during atomization.

In terms of pulmonary delivery of bioactive agents to the systemiccirculation via nebulization, recent research has focused on the use ofportable hand-held ultrasonic nebulizers, also referred to as meteredsolutions. These devices, generally known as single-bolus nebulizers,aerosolize a single bolus of medication in an aqueous solution with aparticle size efficient for deep lung delivery in one or two breaths.These devices fall into three broad categories. The first categorycomprises pure piezoelectric single-bolus nebulizers such as thosedescribed by Mütterlein, et. al., (J. Aerosol Med. 1988; 1:231). Inanother category, the desired aerosol cloud may be generated bymicrochannel extrusion single-bolus nebulizers such as those describedin U.S. Pat. No. 3,812,854. Finally, a third category comprises devicesexemplified by Robertson, et. al., (WO 92/11050) which describes cyclicpressurization single-bolus nebulizers. Each of the aforementionedreferences is incorporated herein in their entirety. Most devices aremanually actuated, but some devices exist which are breath actuated.Breath actuated devices work by releasing aerosol when the device sensesthe patient inhaling through a circuit. Breath actuated nebulizers mayalso be placed in-line on a ventilator circuit to release aerosol intothe air flow which comprises the inspiration gases for a patient.

Regardless of which type of nebulizer is employed, it is an advantage ofthe present invention that biocompatible nonaqueous compounds may beused as suspension mediums. Preferably, they will be able to formaerosols upon the application of energy thereto. In general, theselected suspension medium should be biocompatible (i.e. relativelynon-toxic) and non-reactive with respect to the suspended perforatedmicrostructures comprising the bioactive agent. Preferred embodimentscomprise suspension media selected from the group consisting offluorochemicals, fluorocarbons (including those substituted with otherhalogens), perfluorocarbons, fluorocarbon/hydrocarbon diblocks,hydrocarbons, alcohols, ethers, or combinations thereof. It will beappreciated that, the suspension medium may comprise a mixture ofvarious compounds selected to impart specific characteristics.

In accordance with the teachings herein, the suspension media maycomprise any one of a number of different compounds includinghydrocarbons, fluorocarbons or hydrocarbon/fluorocarbon diblocks. Ingeneral, the contemplated hydrocarbons or highly fluorinated orperfluorinated compounds may be linear, branched or cyclic, saturated orunsaturated compounds. Conventional structural derivatives of thesefluorochemicals and hydrocarbons are also contemplated as being withinthe scope of the present invention as well. Selected embodimentscomprising these totally or partially fluorinated compounds may containone or more hetero-atoms and/or atoms of bromine or chlorine.Preferably, these fluorochemicals comprise from 2 to 16 carbon atoms andinclude, but are not limited to, linear, cyclic or polycyclicperfluoroalkanes, bis(perfluoroalkyl)alkenes, perfluoroethers,perfluoroamines, perfluoroalkyl bromides and perfluoroalkyl chloridessuch as dichlorooctane. Particularly preferred fluorinated compounds foruse in the suspension medium may comprise perfluorooctyl bromide C₈F₁₇Br(PFOB or perflubron), dichlorofluorooctane C₈F₁₆Cl2, and thehydrofluoroalkane perfluorooctyl ethane C₈F₁₇C₂H₅ (PFOE). With respectto other embodiments, the use of perfluorohexane or perfluoropentane asthe suspension medium is especially preferred.

More generally, exemplary fluorochemicals which are contemplated for usein the present invention generally include halogenated fluorochemicals(i.e. C_(n)F_(2n+1)X, XC_(n)F_(2n)X, where n=2-10, X=Br, Cl or I) and,in particular, 1-bromo-F-butane n-C₄F₉Br, 1-bromo-F-hexane (n-C₆F₁₃Br),1-bromo-F-heptane (n-C₇F₁₅Br), 1,4-dibromo-F-butane and1,6-dibromo-F-hexane. Other useful brominated fluorochemicals aredisclosed in U.S. Pat. No. 3,975,512 to Long and are incorporated hereinby reference. Specific fluorochemicals having chloride substituents,such as perfluorooctyl chloride (n-C₈F₁₇Cl), 1,8-dichloro-F-octane(n-ClC₈F₁₆Cl), 1,6-dichloro-F-hexane (n-ClC₆F₁₂Cl), and 1,4-dichloro-F-butane (n-ClC₄F₈Cl) are also preferred.

Fluorocarbons, fluorocarbon-hydrocarbon compounds and halogenatedfluorochemicals containing other linkage groups, such as esters,thioethers and amines are also suitable for use as suspension media inthe present invention. For instance, compounds having the generalformula, C_(n)F_(2n+1)OC_(m)F_(2m+1), orC_(n)F_(2n+1)CH═CHC_(m)F_(2m+1), (as for example C₄F₉CH═CHC₄F₉ (F-44E),i-C₃F₉CH═CHC₆F₁₃ (F-i36E), and C₆F₁₃CH═CHC₆F₁₃ (F-66E)) where n and mare the same or different and n and m are integers from about 2 to about12 are compatible with teachings herein. Usefulfluorochemical-hydrocarbon diblock and triblock compounds include thosewith the general formulas C_(n)F_(2n+1)—C_(m)H_(2m+1) andC_(n)F_(2n+1)C_(m)H_(2m+1), where n=2−12; m=2−16 orC_(p)H_(2p+1)—C_(n)F_(2n)—C_(m)H_(2m+1), where p=1−12, m=1−12 andn=2−12. Preferred compounds of this type include C₈F₁₇C₂H₅, C₆F₁₃C₁₀H₂₁,C₈F₁₇C₈H₁₇, C₆F₁₃CH═CHC₆H₁₃ and C₈F₁₇CH═CHC₁₀H₂₁. Substituted ethers orpolyethers (i.e. XC_(n)F_(2n)OC_(m)F_(2m)X, XCFOC_(n)F_(2n)OCF₂X, wheren and m=1−4, X=Br, Cl or I) and fluorochemical-hydrocarbon etherdiblocks or triblocks (i.e. C_(n)F_(2n+1)—O—C_(m)H_(2m+1), where n=2−10;m=2−16 or C_(p)H_(2p+1)—O—C_(n)F_(2n)—O—C_(m)H_(2m+1), where p=2-12,m=1−12 and n=2-12) may also used as well asC_(n)F_(2n+1)O—C_(m)F_(2m)OC_(p)H_(2p+1), wherein n, m and p are from1-12. Furthermore, depending on the application, perfluoroalkylatedethers or polyethers may be compatible with the claimed dispersions.

Polycyclic and cyclic fluorochemicals, such as C₁₀F₁₈ (F-decalin orperfluorodecalin), perfluoroperhydrophenanthrene,perfluorotetramethylcyclohexane (AP-144) and perfluoro n-butyldecalinare also within the scope of the invention. Additional usefulfluorochemicals include perfluorinated amines, such as F-tripropylamine(“FTPA”) and F-tributylamine (“FTBA”). F4-methyloctahydroquinolizine(“FMOQ”), F-N-methyl-decahydroisoquinoline (“FMIQ”),F-N-methyldecahydroquinoline (“FHQ”), F-N-cyclohexylpyrrolidine (“FCHP”)and F-2-butyltetrahydrofuran (“FC-75” or “FC-77”). Still other usefulfluorinated compounds include perfluorophenanthrene,perfluoromethyldecalin, perfluorodimethylethylcyclohexane,perfluorodimethyldecalin, perfluorodiethyldecalin,perfluoromethyladamantane, perfluorodimethyladamantane. Othercontemplated fluorochemicals having nonfluorine substituents, such as,perfluorooctyl hydride, and similar compounds having different numbersof carbon atoms are also useful. Those skilled in the art will furtherappreciate that other variously modified fluorochemicals are encompassedwithin the broad definition of fluorochemical as used in the instantapplication and suitable for use in the present invention. As such, eachof the foregoing compounds may be used, alone or in combination withother compounds to form the stabilized dispersions of the presentinvention.

Additional exemplarly fluorocarbons, or classes of fluorinatedcompounds, that may be useful as suspension media include, but are notlimited to, fluoroheptane, fluorocycloheptane fluoromethylcycloheptane,fluorohexane, fluorocyclohexane, fluoropentane, fluorocyclopentane,fluoromethylcyclopentane, fluorodimethylcyclopentanes,fluoromethylcyclobutane, fluorodimethylcyclobutane,fluorotrimethylcyclobutane, fluorobutane, fluorocyclobutane,fluoropropane, fluoroethers, fluoropolyethers and fluorotriethylamines.Such compounds are generally environmentally sound and are biologicallynon-reactive.

While any liquid capable of producing an aerosol upon the application ofenergy may be used in conjunction with a nebulizer, the selectedsuspension medium will preferably have a vapor pressure less than about5 atmospheres and more preferably less than about 2 atmospheres. Unlessotherwise specified, all vapor pressures recited herein are measured at25° C. In other embodiments, preferred suspension media compounds willhave vapor pressures on the order of about 5 torr to about 760 torr,with more preferable compounds having vapor pressures on the order offrom about 8 torr to about 600 torr, while still more preferablecompounds will have vapor pressures on the order of from about 10 torrto about 350 torr. Such suspension media may be used in conjunction withcompressed air nebulizers, ultrasonic nebulizers or with mechanicalatomizers to provide effective ventilation therapy. Moreover, morevolatile compounds may be mixed with lower vapor pressure components toprovide suspension media having specified physical characteristicsselected to further improve stability or enhance the bioavailability ofthe dispersed bioactive agent.

Other embodiments of the present invention directed to nebulizers willcomprise suspension media that boil at selected temperatures underambient conditions (i.e. 1 atm). For example, preferred embodiments willcomprise suspension media compounds that boil above 0° C., above 5° C.,above 10° C., above 15°, or above 20° C. In other embodiments, thesuspension media compound may boil at or above 25° C. or at or above 30°C. In yet other embodiments, the selected suspension media compound mayboil at or above human body temperature (i.e. 37° C.), above 45° C., 55°C., 65° C., 75° C., 85° C. or above 100° C.

F(v). Direct Administration

Along with MDIs and nebulizers, it will be appreciated that thestabilized dispersions of the present invention may be used toadminister bioactive agents to a variety of target sites using variousroutes of administration. For example, the disclosed compositions may bedelivered directly to the lungs in conjunction with liquid doseinstillation (LDI) techniques. Alternatively, the stabilized dispersionscould be effectively delivered to mucosal surfaces in the nasal passagesusing a nasal pump, spray bottle or atomizer. In yet other embodiments,the disclosed dispersions could be administered to a target site (e.g.intramuscularly or intradermally) using conventional injections orthrough the use of needleless injectors employing compressed gases. Thelatter are particularly preferred in the case of needleless inoculation.Still other embodiments are directed to the topical delivery of thedispersions to target sites such as the eye or the ear or, morepreferably, mucosal surfaces such as those in the urogenital tract orthe gastrointestinal tract. Such techniques may further employionophoresis to enhance penetration of the incorporated bioactive agent.In any event, the stabilized dispersions provide for excellent dosereproducibility while preserving the activity of the incorporated agent.

Those skilled in the art will appreciate that suspension mediacompatible with the aforementioned delivery techniques are similar tothose set forth above for use in conjunction with nebulizers. That is,the stabilized dispersions will preferably comprise suspension mediaselected from the group consisting of fluorochemicals, fluorocarbons(including those substituted with other halogens), perfluorocarbons,fluorocarbon/hydrocarbon diblocks, hydrocarbons, alcohols, ethers, orcombinations thereof. More particularly, for the purposes of the presentapplication, compatible suspension media for such delivery techniquesshall be equivalent to those enumerated above in conjunction with use innebulizers. In particularly preferred embodiments the selectedsuspension medium shall comprise a fluorochemical that is liquid underambient conditions.

It should be further be appreciated that liquid dose instillationinvolves the direct administration of a stabilized dispersion to thelung. In this regard, direct pulmonary administration of bioactivecompounds is particularly effective in the treatment of disordersespecially where poor vascular circulation of diseased portions of alung reduces the effectiveness of intravenous drug delivery. Withrespect to LDI the stabilized dispersions are preferably used inconjunction with partial liquid ventilation or total liquid ventilation.Moreover, the present invention may further comprise introducing atherapeutically beneficial amount of a physiologically acceptable gas(such as nitric oxide or oxygen) into the pharmaceutical microdispersionprior to, during or following administration.

For LDI, the dispersions of the present invention may be administered tothe lung using a pulmonary delivery conduit. Those skilled in the artwill appreciate the term “pulmonary delivery conduit”, as used herein,shall be construed in a broad sense to comprise any device or apparatus,or component thereof, that provides for the instillation oradministration of a liquid in the lungs. In this respect a pulmonarydelivery conduit or delivery conduit shall be held to mean any bore,lumen, catheter, tube, conduit, syringe, actuator, mouthpiece,endotracheal tube or bronchoscope that provides for the administrationor instillation of the disclosed dispersions to at least a portion ofthe pulmonary air passages of a patient in need thereof. It will beappreciated that the delivery conduit may or may not be associated witha liquid ventilator or gas ventilator. In particularly preferredembodiments the delivery conduit will comprise an endotracheal tube orbronchoscope.

While the stabilized dispersions may be administered up to thefunctional residual capacity of the lungs of a patient, it will beappreciated that selected embodiments will comprise the pulmonaryadministration of much smaller volumes (e.g. on the order of amilliliter or less). For example, depending on the disorder to betreated, the volume administered may be on the order of 1, 3, 5, 10, 20,50, 100, 200 or 500 milliliters. In preferred embodiments the liquidvolume is less than 0.25 or 0.5 percent FRC. For particularly preferredembodiments, the liquid volume is 0.1 percent FRC or less. With respectto the administration of relatively low volumes of stabilizeddispersions it will be appreciated that the wettability and spreadingcharacteristics of the suspension media (particularly fluorochemicals)will facilitate the even distribution of the bioactive agent in thelung. However, in other embodiments it may be preferable to administerthe suspensions a volumes of greater than 0.5, 0.75 or 0.9 percent FRC.Of course the extraordinary wetting and spreading characteristicsassociated with at least some fluorochemicals makes them particularlycompatible for administration to other mucosal surfaces such as thenasal passages.

With regard to the powders and stabilized dispersions disclosed hereinthose skilled in the art will appreciate that they may be advantageouslysupplied to the physician or other health care professional, in asterile, prepackaged or kit form. More particularly, the formulationsmay be supplied as stable powders or preformed dispersions ready foradministration to the patient. Conversely, they may be provided asseparate, ready to mix components. When provided in a ready to use form,the powders or dispersions may be packaged in single use containers orreservoirs, as well as in multi-use containers or reservoirs. In eithercase, the container or reservoir may be associated with the selectedinhalation or administration device and used as described herein. Whenprovided as individual components (e.g., as powdered microspheres and asneat suspension medium) the stabilized preparations may then be formedat any time prior to use by simply combining the contents of thecontainers as directed. Additionally, such kits may contain a number ofready to mix, or prepackaged dosing units so that the user can thenadminister them as needed.

G. EXAMPLES

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples, are, however, merelyrepresentative of preferred methods of practicing the present inventionand should not be read or interpreted as limiting the scope of theinvention in any manner.

I Preparation of Hollow Porous Particles of HA Peptide by Spray-Drying

Hollow porous HA 110-120 peptide (amino acid residues 110-120 of thehemagglutinin of the influenza virus) particles (PulmoSpheres™) wereprepared by a spray drying technique with a B-191 Mini Spray-Drier(Büchi, Flawil, Switzerland) under the following conditions: aspiration:100%, inlet temperature: 85° C.; outlet temperature: 51° C.; feed pump:10%; N₂ flow: 800 L/hr. The feed was prepared by mixing twopreparations, A and B, immediately prior to spray drying. A 150 meshstainless steel screen was placed in the cyclone exit port to aid withthe collection particles.

Preparation A: 5 g of deionized water was used to dissolve 18 mg of HA110-120 peptide (Chiron Corp., Emeryville, Calif.) and 1 mg ofhydroxyethyl starch (Ajinomoto, Japan).

Preparation B: A fluorocarbon-in-water emulsion stabilized byphospholipid was prepared in the following way. The phospholipid, 0.3 gEPC-100-3 (Lipoid KG, Ludwigshafen, Germany), was homogenized in 33 g ofhot deionized water (T=50 to 60° C.) using an Ultra-Turrax mixer (modelT-25) at 8000 rpm for 2 to 5 minutes (T=60-70° C.). Eight grams ofPerflubron (perfluorooctyl bromide: Atochem, Paris, France) was addeddropwise during mixing. After the fluorocarbon was added, the emulsionwas mixed for at least 4 minutes. The resulting coarse emulsion was thenpassed through a high pressure homogenizer (Avestin, Ottawa, Canada) at18,000 psi for passes.

One eighth of preparation B was separated and added to preparation A.The resulting HA peptide/perflubron emulsion feed solution was fed intothe spray dryer under the conditions described above. The powdercollected in the cyclone, and sieving screen was washed into thecollection jar using perflubron. The HA suspension in perflubron wassubsequently frozen at −60° C. and lyophilized. A free flowing whitepowder was obtained.

II In Vitro Activity of Hollow Porous Particles Containing HA Peptide

The functionality of HA peptide in PulmoSpheres (HA-Pul) to activateantigen presenting cells was compared with neat HA peptide. HA peptidePulmoSpheres from Example I were incubated with sterile PBS at aconcentration of 5 mg/ml (weight of formulation/volume). Serialdilutions of the resultant HA-Pul-PBS solution were added to microwellscontaining M12 antigen presenting cells (1×10⁴/well) and HA specific TcH(2×10⁴/well) in complete RPMI-10% FCS. The TcH cell line bears areporter gene controlled by IL-2 promoter (IL-2/-gal).

After 12 hours incubation at 37° C., the microwell plate wascentrifuged, cells were fixed with paraformaldehyde-glutaraldehyde for 5minutes at 4° C., washed with PBS and X-gal substrate was addedovernight. The number of activated TcH per 500 cells per well werecounted using light microscopy. The total number of activated TcH perwell was estimated by multiplying the total number of cells with thepercentage of blue cells. The results, shown in FIG. 1, demonstrate thepresence of active peptide in the formulation. Comparison with astandard activation curve (HA saline) showed that the concentration ofactive peptide was approximately 50% (wt/wt), which was in agreementwith reverse phase-HPLC measurements.

III HA Peptide PulmoSpheres Mechanism of Action

The requirement for internalization and processing of PulmoSpheremicroparticles containing T cell epitope HA 110-120 (HA-Pul) wasexamined. HA-Pul suspended in perflubron (500 nM/well HA 110-120peptide) were air-dried and incubated with non-fixed or paraformaldehydefixed M12 antigen presenting cells (APC) cells in the presence ofspecific TcH cells in complete RPMI-10% FCS, and compared with HA-Pulsuspended in PBS and neat HA peptide at similar concentrations.Sucrose-purified A/PR/8/34 (H1N1) virus (15 g/ml) was used as thepositive control, since it does that require intracellular processing.Negative controls comprised a formulation of NP 147-155 peptide,non-formulated NP peptide and an irrelevant virus. The number of cellsand culture conditions described in Example II were followed. The cellswere fixed and exposed to an X-gal substrate. The results were expressedas % of activated TcH.

FIG. 2 shows that both fixed and non-fixed APC were able to present neatHA peptide and HA-Pul. In contrast, only live APC were able to presentHA peptide from the viral context. Furthermore, formulated or neat NPpeptide as well as B/Lee virus did not activate the specific TcH. Theresults indicate that internalization and processing of HA-Pul is not aprerequisite for the activation of TcH. Rather, the HA-peptide isreadily released from the PulmoSpheres and binds to MHC class IImolecules (I-E^(d)) on M12 APC, resulting in the engagement of TCR andactivation of TcH. This processing step was observed for neat HA peptideas well as HA-Pul delivered in PBS or perflubron. Moreover, theseresults demonstrate that HA 110-120 peptide formulated in PulmoSpheresand stabilized in perflubron retains its immunogenicity.

IV Preparation of Fluorescent-Labeled Hollow Porous HA Peptide Particlesby Spray-Drying

Hollow porous HA-fluoroscein 110-120 peptide/Texas Red DHPE particleswere prepared by a spray drying technique with a B-191 Mini Spray-Drier(Büchi, Flawil, Switzerland) under the following conditions: aspiration:100%, inlet temperature: 85° C.; outlet temperature: 51° C.; feed pump:10%; N₂ flow: 800 L/hr. The feed was prepared by mixing two solutions Aand B immediately prior to spray drying. A 150 mesh stainless steelscreen was placed in the cyclone exit port to aid with the collectionparticles.

Preparation A: 5 g of deionized water was used to dissolve 20 mg ofHA-fluoroscein 110-120 peptide (Chiron Corp., Emeryville, Calif.) and 1mg of hydroxyethyl starch (Ajinomoto, Japan).

Preparation B: A fluorocarbon-in-water emulsion stabilized byphospholipid was prepared in the following way. The phospholipid, 0.3 gEPC-100-3 (Lipoid KG, Ludwigshafen, Germany), and 0.3 mg fluorescentdye, Texas Red DHPE, (Molecular Probes, Eugene, Oreg., 3 mg) were firstdissolved in chloroform. The chloroform was then removed using a BuchiRotoVap. The E100-3/Texas Red DHPE thin film was then dispersed into 33ml hot deionized water (60 to 70° C.). The surfactants were then furtherprocessed in the aqueous phase using an Ultra-Turrax mixer (model T-25)at 10,000 rpm for approximately 2 minutes (T=50 to 60 C). 8 g ofperflubron (Atochem, Paris, France) was added dropwise during mixing.After the fluorocarbon was added, the emulsion was mixed for at least 4minutes. The resulting coarse emulsion was then passed through a highpressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5passes.

One eighth of preparation B was separated and added to preparation A.The resulting HA-fluoroscein peptide/Texas Red DHPE/Perflubron emulsionfeed solution was fed into the spray dryer under the conditionsdescribed above. The powder collected in the cyclone, and sieving screenwas washed into the collection jar using Perflubron. The HA suspensionin Perflubron was subsequently frozen at −60° C. and lyophilized. A freeflowing fluorescent fuschsia-colored powder was obtained.

V Bioavailability of Fluorescent-Labeled HA PulmoSpheres

A formulation comprising fluoroscein-HA peptide (20% wt/wt) PulmoSpheres(f-HA-Pul) prepared as in Example IV was suspended in perflubron.Metofane anesthetized mice were inoculated intranasally (i.n.) with a 70l volume of f-HA-Pul in perflubron, corresponding to 70 g of peptidedose. Blood samples were collected by ocular bleeding in heparin-treatedtubes, the plasma was separated and the concentration of the peptide wasmeasured by fluorometry. As a control, an intravenous i.v.) inoculationof 70 g of f-HA peptide in 70 l of sterile saline (n=4 for all groups)was used.

FIG. 3 depicts the serum concentration of f-HA peptide over time. Theabsolute bioavailability for the i.n. delivered f-HA peptide wasapproximately 5%, with T_(max) occurring at 20 minutes. Thepharmacokinetic profile differed between the two routes ofadministration, with a continuous logarithmic decay for the i.v.administration and a transient increase followed by an exponential decayin the case of i.n. administration. Elimination of f-HA occurs via urine(not shown), with total clearance by 6 hours.

This Example shows that i.n. administration of T cell epitopes (having amolecular weight of approximately 1.4 kDa) formulated in Pul iscompatible with systemic delivery.

VI Preparation of Hollow Porous Particles of Human IgG by Spray-Drying

Hollow porous Human IgG particles were prepared by a spray dryingtechnique with a B-191 Mini Spray-Drier (Büchi, Flawil, Switzerland)under the following conditions: aspiration: 100%, inlet temperature: 85°C.; outlet temperature: 61° C.; feed pump: 10%; N₂ flow: 800 L/hr. Thefeed was prepared by mixing two solutions A and B immediately prior tospray drying.

Preparation A: 2 g of normal saline (Baxter, Chicago, Ill.) was used todissolve 55 mg of human IgG (Sigma Chemicals. St. Louis, Mo.) and 3.2 mgof hydroxyethyl starch (Ajinomoto, Japan).

Preparation B: A fluorocarbon-in-water emulsion stabilized byphospholipid was prepared in the following way. The phospholipid, 0.415g EPC-100-3 (Lipoid KG, Ludwigshafen, Germany), was homogenized in 40.3g of hot deionized water (T=50 to 60° C.) using an Ultra-Turrax mixer(model T-25) at 8000 rpm for 2 to 5 minutes (T=60-70° C.). 5.2 g ofperflubron (Atochem, Paris, France) was added dropwise during mixing.After the fluorocarbon was added, the emulsion was mixed for at least 4minutes. The resulting coarse emulsion was then passed through a highpressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5passes.

One eighth of preparation B by volume was separated and added topreparation A. The resulting IgG/perflubron emulsion feed solution wasfed into the spray dryer under the conditions described above. Thepowder collected in the cyclone, and sieving screen was washed into thecollection jar using perflubron. The IgG suspension in perflubron wassubsequently frozen at −60° C. and lyophilized. A free flowing whitepowder was obtained. The hollow porous IgG particles had avolume-weighted mean aerodynamic diameter of 2.373±1.88 μm as determinedby a time-of-flight analytical method (Aerosizer, Amherst ProcessInstruments, Amherst, Mass.).

VII In Vitro Activity of Polyclonal Human IgG PulmoSpheres

A formulation of polyclonal human IgG PulmoSpheres (hIgG-Pul) fromExample VI was characterized for activity using a capture hIgG ELISA. A5 mg/mL hIgG-Pul suspension in perflubron was prepared, pipetted intothe wells and air dried. PBS was added to the dried hIgG-Pul and allowedto incubate overnight. The hydrated hIgG-Pul solution was diluted andtransferred to an ELISA plate coated with mouse anti-human k chainmonoclonal antibody in coating buffer (dil. 1:1000, SigmaImmunochemical), and subsequently blocked with PBS containing 15% goatserum for 2 hours at room temperature. The wells were washed and theassay was developed using goat anti-human IgG alkaline phosphataseconjugate (1:1000 in PBS-15% goat serum 0.05% Tween), followed byaddition of pNPP substrate. The optical density (OD) was read using atan automatic plate reader set at −405 nm. hIgG in saline (standard) andhIgG mixed with blank PulmoSpheres were employed as controls to rule outan effect of the lipid on the assay. The blank PulmoSpheres werecomprised of only phospholipid and starch.

FIG. 4 depicts the calibration curves for the hIgG-Pul, hIgG and hIgG+blank PulmoSpheres. The hIgG-Pul formulation was determined to compriseapproximately 20% hIgG by weight. In addition, the hIgG-Pul retained theexpression of k light chain and heavy chain epitopes.

VIII Dissolution Kinetics of HA 110-120 Peptide and Human IqG FromPulmoSphere Formulations

The kinetics of antigen and hIgG release from PulmoSpheres was measuredusing dissolution chambers equipped with 0.2 μm diameter filters, andadapted in 24-well flat bottom cell culture plates. Approximately 3 mgof PulmoSphere powder from Examples I and VI were placed in the lowercompartment of the dissolution chamber and exposed simultaneously tosterile PBS (1.3 ml/well). The plates were placed on a horizontal shaker(30 RPM) at 37° C., to simulate the breathing pattern. 25 μl sampleswere collected from the upper compartment and analyzed by capture ELISAin the case of hIgG (FIG. 5A) or bioassay in the case offluoroscein-labeled HA peptide formulation (f-HA) (FIG. 5B). The resultsfor f-HA were independently confirmed by fluorometry (not shown). Thedissolution kinetics of the hIgG and HA peptide PulmoSphere formulationswere compared with their respective aqueous controls.

The results depicted in FIGS. 5A and 5B and were expressed as percentrelease. A rapid diffusion-controlled release was observed for HApeptide formulation, with no difference between the aqueous control.Complete dissolution occurred within 2 hours. In contrast, a slowererosion-controlled kinetics was observed for the hIgG formulation.Complete dissolution required more than 6 hours as compared with 1 hourfor the aqueous IgG control. The results described herein demonstratethat the dissolution kinetics from PulmoSpheres depends, at least inpart, on the molecular weight of the formulated compound (1.4 kDa and150 kDa, respectively). It will also be appreciated that differences inhydrophilicity or hydrophobicity may have similar effects.

IX Bioavailability of hIgG PulmoSpheres

Human IgG PulmoSpheres (hIgG-Pul) from Example VI were administeredeither intratracheally (20 g hIgG in 20 l of perflubron) to miceanesthetized with ketamine/xylazine, or via the nasal route (70 g hIgGin 70 μl of perflubron) to mice anesthetized with metofane. An identicalvolume of hIgG in sterile PBS was administered intravenously in thecontrol group. The mice were bled at various time intervals and theserum concentration of hIgG was assessed by capture ELISA in all groups(n=3). Absolute bioavailability was determined from the areas under theserum concentration-time curve (AUC) as compared with the i.v. control.AUC values were calculated using the trapezoid rule.

The plamsa hIgG concentration curves are depicted in FIGS. 6A (i.t.),and 6B (i.n.). The absolute bioavailability for the intratrachealdelivery of hIgG-Pul was 27%, and 1.5% for intranasal inoculation. Inboth cases, the T_(max) occurred at approximately 2 days. Westernblotting showed that the molecular weight for the circulating hIgG afterdelivery via the respiratory tract was indistinguishable from the neatmaterial. The hIgG was observed to persist in the circulation more than14 days.

X Antibody Response to hIgG PulmoSpheres Delivered via the TrachealRoute

The humoral response in the blood and bronchoalveolar lavage (BAL) inmice treated with hIgG-PulmoSpheres (hIgG-Pul) from Example VI suspendedin perflubron via intratracheal administration (20 g dose of hIgG). Micewere also treated with the following controls: 20 μg hIgG in salinei.t., 100 μg hIgG in saline i.v. and i.t., 100 μl g in complete Freund'sAdjuvant (CFA) subcutaneous and saline it. Each group was done intriplicate. Blood and BAL were collected 2 weeks after immunization.

The titer of anti-hIgG mouse IgG was measured using ELISA plates coatedwith hIgG or with 0.1% BSA. The wells were blocked with PBS-15% goatserum and incubated for two hours using various dilutions of sera orBAL. After washing, the assay was developed with goat anti-mouse IgGalkaline phosphatase conjugate, followed by the addition of pNPPsubstrate. The optical density (OD) of the plates were analyzed at 550nm using an automatic plate reader and the results were expressed asendpoint dilution titers in the case of serum IgG (FIG. 7A) or mean ODfor BAL IgG (FIG. 7B).

The results show an increased systemic and local humoral responses inmice treated with hIgG-Pul via the intratracheal route, as compared withthe dose/route matched group that received hIgG in saline. Moreover, theresponse was enhanced as compared with mice that received higher dosesof hIgG in saline, via intratracheal or intravenous routes. The titer ofserum antibodies was similar to that measured in mice immunized s.c.with hIgG in CFA. Interestingly, the humoral response did not correlatewith the systemic bioavailability (data not shown), implying theparticipation of local immunity.

XI T Cell Response to hIgG PulmoSpheres Delivered via Tracheal Route

The level of T cell immunity induced in the spleens of mice immunizedwith hIgG-PulmoSpheres (hIgG-Pul) from Example VI suspended inperflubron by the tracheal route. The spleens were dissociated intosingle cell suspensions that were treated with hypotonic buffer toremove the red blood cells. The splenocytes were resuspended in completeRPMI-10% FCS at 4×10⁶ cells/ml and incubated in 24-well flat bottomplates (1 ml/well), in the presence of 6 g/ml of hIgG. After 72 hoursincubation, the supernatants were collected and the concentration ofIL2, IFN- and IL-4 determined by ELISA (Biosource International,Camarillo, Calif.).

The results (FIG. 8) were expressed as mean values of cytokineconcentration among individual mice in each group, and showed enhancedproduction of all three cytokines in mice immunized with hIgG-Pul ascompared with the hIgG saline controls. The production of cytokines bysplenic T cells for the hIgG-Pul treated group was comparable with thatobserved for the i.v. hIgG in saline group. These results stronglysuggest systemic migration of memory T cells primed in the lung.

XII Antibody Response to hIgG PulmoSpheres Delivered Via the Nasal Route

The humoral response of mice that received hIgG via intranasalinstillation (20 g) either formulated as PulmoSpheres (hIgG-Pul) fromExample VI suspended in perflubron or dissolved in saline wascharacterized. Sera was obtained at various time intervals afterimmunization and the titer of specific mouse IgG raised against the hIgGwas measured using the ELISA procedure described at Example X. Theresults (FIG. 9) were expressed as mean endpoint titers (n=3), andshowed that the kinetics of onset was faster, the magnitude was higherand the intersubject reproducibility of immune responses was lower inmice treated with hIgG-Pul as compared to saline.

XIII Antibody Response to hIgG PulmoSpheres Delivered Via PeritonealRoute

The humoral response of mice treated with hIgG-Pulmospheres (hIgG-Pul)from Example VI suspended in perflubron via the peritoneal (i.p.) route(100 g dose of hIgG). Mice were also treated i.p. with 100 μg hIgG inthe following controls: in saline, in a multilamellardipalmitoylphospahtidylcholine (DPPC) liposome saline solution (+mllip), in a unilamellar DPPC liposome saline solution (+ul lip) and in ablank PulmoSphere saline solution (+empty Pul). An additional controlgroup of blank PulmoSphere solution devoid of hIgG was also tested. Theparticle median diameter of ml lip (>10 μm) and ul lip (90 nm) weredetermined using a laser light scattering technique. Each group was donein triplicate. The IgG humoral immune response in sera, at 7 and 14 dayswas measured using the same ELISA technique described in Example X.

The results were expressed as means of endpoint titers and showed aconsistent increase in antibody titers for animals that were inoculatedwith hIgG-Pul. More particularly FIGS. 10A and 10B show endpoint titersat 7 and 14 days respectively. hIgG added to empty Pul induced titerssimilar to hIgG in saline. Furthermore, addition of either DPPC liposomepreparation to hIgG did not restore the increased immunity observed withhIgG-Pul. Thus, these results demonstrate that an: (1) enhanced immunityhIgG-Pul is not a route dependent phenomenon (see Examples X and XII);(2) formulation of hIgG-Pul is a prerequisite for the enhancedimmunogenicity of hIgG; and (3) DPPC or other components of Pul do nothave an independent adjuvant effect. Moreover, these results elucidatethe importance of the route of delivery as well as other factorsresponsible for the enhanced immunity elicited by hIgG-Pul.

XIV Preparation of Hollow Porous Particles of Influenza Virus A/WSN/32(H1N1) by Spray-Drying

Hollow porous Influenza Virus (A/WSN/32 H1N1), which comprises arelatively complex enveloped virus comprising 8 structural proteincomplexes and 8 negatively charged RNA segments, were successfullyincorporated in microparticles prepared by a spray drying technique witha B-191 Mini Spray-Drier (Büchi, Flawil, Switzerland) under thefollowing conditions: aspiration: 100%, inlet temperature: 85° C.;outlet temperature: 61° C.; feed pump: 10%; N₂ flow: 800 L/hr. The feedwas prepared by mixing two preparations A and B immediately prior tospray drying. Prior to formulation, the virus was live and had beenpurified by sucrose-gradient centrifugation.

Preparation A: Weighed 1 mg hydroxyethyl starch (Ajinomoto, Japan) andtransferred to tube containing 0.6 mg Influenza Virus in saline.

Preparation B: A fluorocarbon-in-water emulsion stabilized byphospholipid was prepared in the following way. The phospholipid, 0.111g EPC-100-3 (Lipoid KG, Ludwigshafen, Germany), was homogenized in 20 gof hot deionized water (T=50 to 60° C.) using an Ultra-Turrax mixer(model T-25) at 8000 rpm for 2 to 5 minutes (T=60-70° C.). 4.4 g ofperflubron (Atochem, Paris, France) was added dropwise during mixing.After the fluorocarbon was added, the emulsion was mixed for at least 4minutes. The resulting coarse emulsion was then passed through a highpressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5passes.

One eighth of preparation B by volume was separated and added topreparation A. The resulting Influenza Virus/perflubron emulsion feedsolution was fed into the spray dryer under the conditions describedabove. The powder collected in the cyclone, and the sieving screen waswashed into the collection jar using perflubron. The Influenza Virussuspension in perflubron was subsequently frozen at −60° C. andlyophilized. A free flowing white powder was obtained.

XV In Vitro Activity of Influenza Virus A/WSN/32 (H1N1) PulmoSpheres

The incorporation of live viral antigen into spray-dried particles wascharacterized using the following technique: Influenza Virus A/WSN/32(H1N1) PulmoSpheres (WSN-Pul) from Example XIV were dissolved in sterilePBS at a concentration of 5 mg/ml for 6 hours at 40° C. The hydratedWSN-Pul was then incubated at various dilutions with non-fixed orparaformaldehyde-fixed M12 antigen presenting cells (APC) for 1 hour at37° C., in 96-well plates. After antigen pulsing, the APCs were washedand incubated for four hours with TcH. The formaldehyde-glutaraldehydefixed cells were incubated with X-gal substrate, and positive cells werecounted.

Results were expressed as percent activated TcH (FIG. 11A). Variousconcentrations of sucrose-purified live WSN virus were used as controls(FIG. 11B). The WSN-Pul formulation was determined to containapproximately 5% influenza virus by weight. Only the unfixed APC's couldactivate the virus, indicating that the antigens had not degraded.Titration of infectious virus was determined by MDCK (Madine Darbykidney carcinoma cells) assay (FIG. 11C), and showed that approximately1% of the total virus was still able to infect and replicate in thepermissive cells. Together, these results demonstrate successfulincorporation of relatively large influenza virus antigens inPulmoSphere powders.

XVI Antibody Response to Influenza Virus A/WSN/32 (H1N1) PulmoSpheresDelivered Via Nasal Route

The induction of virus-specific IgG antibody response against WSN virusafter intranasal inoculation of BALB/c mice with an Influenza VirusA/WSN/32 (H1N1) PulmoSphere (WSN-Pul) formulation containing 5 g ofvirus and 2×10³ TCID₅₀ of live virus (1% of the total antigen loadcorresponding to the amount of live virus) was measured. Control micewere immunized mice with 2×10³ TCID₅₀ live virus (corresponding to 0.05g of total virus) or UV-killed WSN virus (5 g). Sera from mice treatedwith hIgG was used as negative control. The antibody response wasmeasured in sera using the following ELISA technique: wells were coatedwith sucrose purified WSN virus in coating buffer, blocked withnon-mammalian proteins (SeraBlock) and incubated with serial dilutionsof serum samples. The samples were washed, and the assay was developedwith biotin conjugated rat anti-mouse mAb followed bystrptavidin-alkaline phosphatase and pNPP substrate. The results wereexpressed as geometrical means of reciprocal endpoint titers. The numberof mice per inoculation group was three.

The results depicted in FIG. 12 show the induction of high titers of IgGantibodies in mice immunized with WSN-Pul or live WSN virus in saline(WSN/lo) at 7 and 14 days. In contrast, only small titers of specificIgG were detected in mice immunized with killed virus in saline.

XVII T Cell Response to Influenza Virus A/WSN/32 (H1N1) PulmoSpheresDelivered Via Nasal Route

The T cell response was defined in terms of virus and epitope-specificcytokine production of lymphocytes from mice immunized as describedabove (Example XVI). The induction of T-cell response after intranasalinoculation of BALB/c mice with a Influenza Virus A/WSN/32 (H1N1)PulmoSpheres (WSN-Pul) formulation containing 5 g of virus and 2×10³TCID₅₀ of live virus (1% of the total antigen load corresponding to theamount of live virus) was measured. Control mice were immunized micewith 2×10³ TCID₅₀ live virus (corresponding to 0.05 g of total virus) orUV-killed WSN virus (5 g). The antigens examined were sucrose-purifiedWSN virus, HA 110-120 peptide and NP 147-155 peptide. An untreatedsaline group was included as control.

Peripheral blood mononuclear cells (PBMC) were isolated from blood atday 10 after immunization, by Ficoll gradient centrifugation. Variousnumbers of responder cells were incubated in nitrocellulose/anti-IFN oranti-IL-4 (PharMingen) ELISPOT plates (Millipore) at 3×10⁵ cells/well incomplete RPMI-10% FCS. Stimulator cells (mytomicin treated splenocytes,5×10⁵/well), antigens and human rIL-2 (20 U/ml) were added and theplates were co-incubated for 48 hours. The cells were then washed withPBS-0.05% Tween, anti-cytokine antibodies (PharMingen) were incubatedovernight and the assay was developed using HRP-streptavidin conjugatefollowed by insoluble substrate (Vector Laboratories). The assay wasstopped with water, the wells were air-dried and the spots were countedusing a stereomicroscope.

The results were expressed as the frequency of specific cells thatproduce IFN- or IL-4/10⁶ PBMC, after subtracting the background signal.The background was reproducibly below 6/10⁶. PBMC were pooled from themice in each group. The results in FIGS. 13A, 13B and 13C show thatvaccination with WSN-Pul and WSN virus generally induced HA-, NP- andWSN-specific T cells producing IFN- and IL-4. In contrast, immunizationwith killed virus induced predominantly IL-4 producing T cells.Moreover, the immunization with killed virus induced an enhancedsubpopulation of IL-4 producing Tc2 cells, specific for the NP 147-155peptide. These data indicate that the T cell response provoked by thelive control and formulated virus (i.e. comprising live and killedvirus) was more effective the response provoked by the killed viruscontrol corresponding to typical conventional vaccines.

XVIII Protection Against Infectious Challenge of Mice Immunized WithInfluenza Virus A/WSN/32 (H1N1) PulmoSpheres Delivered Via Nasal Route

Mice immunized as described in Example XVII were challenged at threeweeks after immunization with 1.2×10⁶ influenza virus delivered via thenasal route. The protection in terms of virus shedding and variation ofbody weight were defined at day 4 after the challenge. The results areshown in FIGS. 14A and 14B.

Measurement of virus titers in the nasal wash was determined bytitrating the live virus in the MDCK assays. Results showed the absenceof infectious virus in mice previously immunized with Influenza VirusA/WSN/32 (H1N1) PulmoSpheres (WSN-Pul) or control live WSN virus (FIG.14A). Mice immunized with UV killed WSN virus or naive mice displayedsignificant titers of influenza virus in the nasal wash. In addition,the mice immunized with WSN-Pul or WSN virus (low dose of live virus)retained their body weight following the challenge (FIG. 14B). Whereasthe non-immunized mice and those immunized with UV killed WSN virusdisplayed significant reduction of body weight followed by death (⅔ ineach group by day 7). These results demonstrated that the WSN-Pul canprovide effective vaccination efficiency upon mucosal delivery.

XIX Preparation of Hollow Porous Particles of TA7 Retrovirus bySpray-Drying

Hollow porous TA7 Retrovirus particles were prepared by a spray dryingtechnique with a B-191 Mini Spray-Drier (Büchi, Flawil, Switzerland)under the following conditions: aspiration: 100%, inlet temperature: 85°C.; outlet temperature: 61° C.; feed pump: 10%; N₂ flow: 800 L/hr. Thefeed was prepared by mixing two solutions A and B immediately prior tospray drying.

Preparation A: 2 g of deionized water was used to dissolve 1 mg of TA7Retrovirus.

Preparation B: A fluorocarbon-in-water emulsion stabilized byphospholipid was prepared in the following way. The phospholipid, 0.3 gEPC-100-3 (Lipoid KG, Ludwigshafen, Germany), was homogenized in 16.5 gof hot deionized water (T=50 to 60° C.) using an Ultra-Turrax mixer(model T-25) at 8000 rpm for 2 to 5 minutes (T=60-70° C.). 8.0 g ofperflubron (Atochem, Paris, France) was added dropwise during mixing.After the fluorocarbon was added, the emulsion was mixed for at least 4minutes. The resulting coarse emulsion was then passed through a highpressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5passes.

One eighth of preparation B by volume was separated and added topreparation A. The resulting TA7 Retrovirus/perflubron emulsion feedsolution was fed into the spray dryer under the conditions describedabove. The powder collected in the cyclone, and sieving screen waswashed into the collection jar using Perflubron. The TA7 Retrovirussuspension in perflubron was subsequently frozen at −60° C. andlyophilized. A free flowing white powder was obtained.

XX In Vitro Activity of TA7 Retrovirus Spray-Dried Particles

The activity of TA7 Retrovirus following incorporation into thespray-dried particles prepared in Example XIX was examined. Spray-driedTA7 Retrovirus particles were dissolved in saline and applied to Helacells for 1 hour. 24 h hours post inoculation, the cells were thenassayed for transgenic expression using β-gal. No difference wasobserved between the neat and spray-dried TA7 Retrovirus I particles.These results demonstrate that the TA7 Retrovirus, a relatively largeand complex entity, can be effectively incorporated in spray-driedparticles with no apparent loss of activity.

XXI Preparation of Hollow Porous Particles of Bovine Gamma Globulin bySpray-Drying

Hollow porous bovine gamma globulin (BGG) particles were prepared by aspray drying technique with a B-191 Mini Spray-Brier (Büchi, Flawil,Switzerland) under the following conditions: aspiration: 100%, inlettemperature: 85° C.; outlet temperature: 61° C.; feed pump: 10%; N₂flow: 800 L/hr. The feed was prepared by mixing two solutions A and Bimmediately prior to spray drying.

Preparation A: 21 g of 0.2% saline solution was used to dissolve 0.6 gof BGG (CalBiochem San Diego, Calif.), 0.42 g Lactose (Sigma Chemicals,St. Louis, Mo.) and 25 mg of Pluronic F-68, NF grade (BASF, Parsippany,N.Y.).

Preparation B: A fluorocarbon-in-water emulsion stabilized byphospholipid was prepared in the following way. The phospholipid, 1.02 gEPC-100-3 (Lipoid KG, Ludwigshafen, Germany), was homogenized in 30 g ofhot deionized water (T=50 to 60° C.) using an Ultra-Turrax mixer (modelT-25) at 8000 rpm for 2 to 5 minutes (T=60-70° C.). 35 g of F-decalin(Air Products, Allentown, Pa.) was added dropwise during mixing. Afterthe fluorocarbon was added, the emulsion was mixed for at least 4minutes. The resulting coarse emulsion was then passed through a highpressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5passes.

Preparations A and B were combined and fed into the spray-dryer underthe conditions described above. A free flowing white powder wascollected at the cyclone separator. The hollow porous particles had avolume-weighted mean aerodynamic diameter of 1.27±1.42 μm as determinedby a time-of-light analytical method (Aerosizer, Amherst ProcessInstruments, Amherst, Mass.).

XXII Andersen Cascade Impactor Results for Bovine Gamma Globulin MDIFormulations

The inhalation properties of a metered dose inhaler (MDI) formulatedwith hollow porous particles of BGG was prepared according to ExampleXXI was assessed using an Andersen Cascade impactor. 83 mg of the hollowporous BGG particles was weighed a into 10 ml aluminum can, and dried ina vacuum oven under the flow of nitrogen for 3-4 hours at 40° C. The canwas crimp sealed using a DF31/50act 50 I valve (Valois of America,Greenwich, Conn.) and filled with 9.64 g HFA-134a (DuPont, Wilmington,Del.) propellant by overpressure through the stem.

Upon actuation of the apparatus, a fine particle fraction of 61% andfine particle dose of 68 μg were observed (FIG. 15). The instant exampleillustrates that a relatively large bioactive agent such as BGG can beformulated and effectively delivered from a MDI.

Those skilled in the art will further appreciate that the presentinvention may be embodied in other specific forms without departing fromthe spirit or central attributes thereof. In that the foregoingdescription of the present invention discloses only exemplaryembodiments thereof, it is to be understood that, other variations arecontemplated as being within the scope of the present invention.Accordingly, the present invention is not limited to the particularembodiments that have been described in detail herein. Rather, referenceshould be made to the appended claims as indicative of the scope andcontent of the invention.

What is claimed is:
 1. A medicament for the modulation of the immunesystem of a subject comprising a plurality of microstructures associatedwith one or more immunoactive agents, wherein said microstructurescomprise at least about 5% w/w of a biocompatible surfactant selectedfrom the group consisting of saturated and unsaturated lipids, nonionicdetergents, nonionic block copolymers, ionic surfactants, cationicsurfactants, biocompatible fluorinated surfactants, and combinationsthereof; and wherein the immune response elicited by the composition ofthe present invention is greater than the immune response provoked byintravenous or intraperitoneal administration of the same antigensolubilized or suspended in an aqueous carrier.
 2. The medicament ofclaim 1, wherein said microstructures further comprise at least onepenetration enhancing excipient selected from the group consisting of:chelating agents, surfactants, fatty acids, bile salts, and combinationsthereof.
 3. The medicament of claim 2, wherein said at least onepenetration enhancing excipient is a short-chain phospholipid with achain length of 10 carbons or less.
 4. The medicament of claim 1,wherein said biocompatible surfactant is selected from the groupconsisting of phospholipids, poloxamers, and combinations thereof. 5.The medicament of claim 4, wherein said phospholipid is selected fromthe group consisting of dilauroylphosphatidylcholine,dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine,disteroylphosphatidylcholine, dibehenoylphosphatidylcholine,diarachidoylphosphatidylcholine, and combinations thereof.
 6. Themedicament of claim 1, wherein said microstructures comprise at leastabout 25% w/w of said biocompatible surfactant.
 7. The medicament ofclaim 1, wherein said microstructures are dispersed in a nonaqueoussuspension medium.
 8. The medicament of claim 7, wherein said nonaqueoussuspension medium comprises a compound selected from the groupconsisting of hydrofluoroalkanes, fluorocarbons, perfluorocarbons,fluorocarbon/hydrocarbon diblocks, hydrocarbons, alcohols, ethers, andcombinations thereof.
 9. The medicament of claim 7, wherein saidnonaqueous suspension medium comprises a compound selected from thegroup consisting of liquid fluorochemicals and hydrofluoroalkanepropellants.
 10. The medicament of claim 1, wherein the mean aerodynamicdiameter of said microstructures is between about 0.5 and about 5 μm.11. The medicament of claim 1, wherein said microstructures have a meangeometric diameter of between about 1 and about 30 μm.
 12. Themedicament of claim 1, wherein said microstructures have a meangeometric diameter of less than about 5 μm.
 13. The medicament of claim1, wherein said microstructures are selected from the group consistingof particulates, microparticulates, perforated microstructures, andcombinations thereof.
 14. The medicament of claim 1, wherein saidmicrostructures are perforated microstructures.
 15. The medicament ofclaim 14, wherein said perforated microstructures comprise hollow porousmicrostructures.
 16. The medicament of claim 1, wherein saidimmunoactive agent is selected from the group consisting of immunoactivepeptides, polypeptides, proteins, carbohydrates, genetic material, andmicrobes.
 17. The medicament of claim 1, wherein said immunoactive agentcomprises a vaccine.
 18. The medicament of claim 17, wherein saidvaccine is selected from the group consisting of inactivated microbes,live attenuated microbes, phages, subunit vaccine proteins, subunitvaccine peptides, subunit vaccine carbohydrates, replicons, viralvectors, plasmids, and combinations thereof.
 19. The medicament of claim1, wherein said modulation of the immune system of a subject comprisesan immune response selected from the group consisting of: eliciting animmune response to a foreign antigen or pathogenic particle; inducinglocalized or systemic passive immunity; stimulating an immune response;and down regulating an immune reaction.
 20. The medicament of claim 1,wherein said modulation of the immune system of a subject comprisesmucosal immunity.
 21. The medicament of claim 1, wherein said medicamentis formulated so as to be capable of being administered to said subjectusing a delivery methodology selected from the group consisting oftopical, intramuscular, transdermal, intradermal, intraperitoneal,nasal, pulmonary, vaginal, rectal, aural, oral or ocular administration.22. A vaccine for eliciting an enhanced immune response in a subjectcomprising a plurality of microstructures associated with one or moreimmunoactive agents, wherein said microstructures comprise at least 5%w/w of a biocompatible surfactant selected from the group consisting ofsaturated and unsaturated lipids, nonionic detergents, nonionic blockcopolymers, ionic surfactants, cationic surfactants, biocompatiblefluorinated surfactants, and combinations thereof, wherein said vaccineis formulated so as to be capable of being administered to therespiratory tract of said subject; and wherein said enhanced immuneresponse is enhanced relative to the immune response elicited by acomparable immunoactive agent delivered via an aqueous carrier in thesubstantial absence of said microstructures.
 23. The vaccine of claim22, wherein said biocompatible surfactant is selected from the groupconsisting of phospholipids, poloxamers, and combinations thereof. 24.The vaccine of claim 23, wherein said phospholipid is selected from thegroup consisting of dilauroylphosphatidylcholine,dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine,disteroylphosphatidylcholine, dibehenoylphosphatidylcholine,diarachidoylphosphatidylcholine, and combinations thereof.
 25. Thevaccine of claim 22, wherein said immunoactive agent is selected fromthe group consisting of inactivated microbes, live attenuated microbes,phages, subunit vaccine proteins, subunit vaccine peptides, subunitvaccine carbohydrates, replicons, viral vectors, plasmids, otherimmunoactive genetic and recombinant materials, and combinationsthereof.
 26. The vaccine of claim 22, wherein said vaccine is compatiblewith administration via a dry powder inhaler.
 27. The vaccine of claim22, wherein said microstructures are dispersed in a nonaqueoussuspension medium.
 28. The vaccine of claim 27, wherein said nonaqueoussuspension medium comprises a compound selected from the groupconsisting of liquid fluorochemicals and hydrofluoroalkane propellants.29. The vaccine of claim 27, wherein said vaccine is administered usinga metered dose inhaler, a nebulizer, an atomizer, a nasal pump, or aspray bottle.
 30. The vaccine of claim 22, wherein said microstructuresare selected from the group consisting of: particulates,microparticulates, and perforated microstructures.
 31. The vaccine ofclaim 22, wherein said microstructures comprise perforatedmicrostructures.
 32. The vaccine of claim 31, wherein said perforatedmicrostructures comprise hollow porous microstructures.
 33. The vaccineof claim 22, wherein the mean aerodynamic diameter of saidmicrostructures is between about 0.5 and about 5 μm.
 34. The vaccine ofclaim 22, wherein said microstructures have a mean geometric diameter ofbetween about 1 and about 30 μm.
 35. The vaccine of claim 22, whereinsaid microstructures have a mean geometric diameter of less than about 5μm.
 36. The vaccine of claim 22, wherein said elicited immune responsecomprises mucosal and/or systemic immunity.
 37. The vaccine of claim 22,wherein said microstructures further comprise an immunogenicitymodifying excipient.
 38. The vaccine of claim 37, wherein saidimmunogenicity modifying excipient is selected from the group consistingof mannans, cell-binding polysaccharides, cofactors, cytokines, andcombinations thereof.
 39. The vaccine of claim 22, wherein said immuneresponse is enhanced by at least about 25% relative to the immuneresponse elicited by a comparable immunoactive agent delivered via anaqueous carrier in the substantial absence of said microstructures. 40.A method for providing enhanced active immunization comprisingadministering to a patient a therapeutically or prophylacticallyeffective amount of a medicament that comprises a plurality ofmicrostructures associated with one or more vaccines, wherein saidmicrostructures comprise at least about 5% w/w of a biocompatiblesurfactant selected from the group consisting of saturated andunsaturated lipids, nonionic detergents, nonionic block copolymers,ionic surfactants, cationic surfactants, biocompatible fluorinatedsurfactants, and combinations thereof; and wherein said enhanced activeimmunization is enhanced relative to the immune response elicited bycomparable vaccines delivered via an aqueous carrier in the substantialabsence of said microstructures.
 41. The method of claim 40, whereinsaid biocompatible surfactant is selected from the group consisting ofphospholipids, poloxamers, and combinations thereof.
 42. The method ofclaim 40, wherein said vaccine is selected from the group consisting ofinactivated microbes, live attenuated microbes, phages, subunit vaccineproteins, subunit vaccine peptides, subunit vaccine carbohydrates,replicons, viral vectors, plasmids, other immunoactive genetic andrecombinant materials, and combinations thereof.
 43. The method of claim40, wherein said medicament is formulated so as to be capable of beingdelivered to or via the respiratory tract.
 44. The method of claim 40,wherein said microstructures further comprise an immunogenicitymodifying excipient.
 45. The method of claim 44, wherein saidimmunogenicity modifying excipient is selected from the group consistingof mannans, cell-binding polysaccharides, cofactors, cytokines, andcombinations thereof.
 46. A method for providing enhanced passiveimmunization comprising administering to a patient a therapeutically orprophylactically effective amount of a medicament that comprises aplurality of microstructures associated with one or more immunoglobulinsor immunoglobulin-like molecules, wherein said microstructures compriseat least about 5% w/w of a biocompatible surfactant selected from thegroup consisting of saturated and unsaturated lipids, nonionicdetergents, nonionic block copolymers, ionic surfactants, cationicsurfactants, biocompatible fluorinated surfactants, and combinationsthereof; and wherein said enhanced passive immunization is enhancedrelative to the immune response elicited by comparable immunoglobulinsor immunoglobulin-like molecules delivered via an aqueous carrier in thesubstantial absence of said microstructures.
 47. The method of claim 46,wherein said biocompatible surfactant is selected from the groupconsisting of phospholipids, poloxamers, and combinations thereof. 48.The method of claim 46, wherein said medicament is formulated so as tobe delivered to or via the respiratory tract.
 49. A method for treatingan autoimmune disease or disorder comprising administering to a patienta therapeutically or prophylactically effective amount of a medicamentthat comprises a plurality of microstructures associated with one ormore immunomodulators, wherein said microstructures comprise at leastabout 5% w/w of a biocompatible surfactant selected from the groupconsisting of saturated and unsaturated lipids, nonionic detergents,nonionic block copolymers, ionic surfactants, cationic surfactants,biocompatible fluorinated surfactants, and combinations thereof; andwherein said treatment for an autoimmune disease or disorder is enhancedrelative to the treatment elicited by comparable immunomodulatorsdelivered via an aqueous carrier in the substantial absence of saidmicrostructures.
 50. The method of claim 49, wherein saidimmunomodulators are selected from the group consisting of: foreignantigens, self-antigens, antigen epitope peptides, immunoglobulins,autoimmune related-ligands, cytokines, and combinations thereof.
 51. Themethod of claim 49, wherein said biocompatible surfactant is selectedfrom the group consisting of phospholipids, poloxamers, and combinationsthereof.
 52. The method of claim 49, wherein said medicament isformulated so as to be capable of being delivered to or via therespiratory tract.